Reagents and methods for detecting neisseria gonorrhoeae

ABSTRACT

This invention provides compositions and methods for detecting  Neisseria gonorrhoeae  in a sample. This invention also provides related reaction mixtures, kits, systems, and computers.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.11/017,476 filed Dec. 17, 2004, the disclosure of which is incorporatedby reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to the fields of molecular biology andnucleic acid chemistry. The invention provides methods and reagents fordetecting pathogens, such as Neisseria gonorrhoeae and accordingly, alsorelates to the fields of medical diagnostics and prognostics.

BACKGROUND OF THE INVENTION

The genus Neisseria consists of Gram-negative aerobic bacteria includingthe human pathogen N. gonorrhoeae, which is the causative agent ofgonorrhea. N. gonorrhoeae infections, which have a high prevalence andlow mortality, are generally acquired by sexual contact and typicallyaffect mucous membranes of the urethra in males and the endocervix infemales. However, the infection may also spread to other tissues. Forexample, a genital infection in males can ascend the urethra and producesymptoms of prostatitis, whereas in females, an N. gonorrhoeae infectionof the cervix may spread to the fallopian tubes and ultimately causesterility among other conditions, if untreated. The pathogenic mechanismof N. gonorrhoeae involves the attachment of the bacterium tononciliated epithelial cells via pili. The mechanism also includes theproduction of endotoxin and IgA proteases.

Co-infection of N. gonorrhoeae and Chlamydia trachomatis is frequentlyobserved. Both infections are two known causes of ectopic pregnancy andcan also lead to infertility if untreated. They are also known causes ofthe acute clinical syndromes of mucopurulent cervicitis and pelvicinflammatory disease. Therefore, the detection of N. gonorrhoeae and C.trachomatis infections, which can be asymptomatic, especially infemales, is of consequence to individuals in need of treatment and tobroader populations at risk of acquiring and further propagating theinfections.

The detection and identification of bacterial infections hastraditionally been accomplished by pure culture isolation anddetermination procedures that make use of knowledge of specimen source,growth requirements, visible growth features, microscopic morphology,staining reactions, and biochemical characteristics. For example,pre-existing methods of detecting and identifying N. gonorrhoeaeinfections, include Gram-staining, culturing on selective agar media,and cytochrome oxidase and carbohydrate utilization testing. Serologicalassays, including coagglutination and fluorescent antibody staining havealso been described for the detection of N. gonorrhoeae. Culture-basedmethods, while relatively sensitive, are generally slow to perform,often including overnight incubation, and are labor intensive. TheGram-stain and antibody-based tests typically provide results in lessthan one hour, but are generally of lower sensitivity than culture-basedmethods.

The use of specific polynucleotide sequences as probes for therecognition of infectious agents is one alternative to problematicimmunological identification assays and other preexisting methodologies.For example, nucleic acid probes complementary to targeted nucleic acidsequences have been used in hybridization procedures, such as Southernblots and dot blots, to detect the target nucleic acid sequence. Many ofthese hybridization procedures have depended on the cultivation and/orenrichment of the organism and, thus, are unsuitable for rapiddiagnosis. The advent of techniques for the rapid amplification ofspecific nucleic acid sequences, such as the polymerase chain reactionamong many others, have provided a mechanism to use sequence specificprobes directly on clinical specimens, thereby eliminating enrichmentand in vitro culturing of the pathogen prior to performing thehybridization assay. Thus, amplification-based hybridization assays canprovide simple and rapid diagnostic techniques for the detection ofpathogens in clinical samples.

Many probes used to date lack sufficient specificity to differentiatebetween pathogenic agents having highly homologous nucleic acidsequences, such as N. gonorrhoeae, N. meningitidis, and the like. Thiscan lead to biased assay results, including false positives. Oneconsequence of such misdiagnosis may be the administration of aninappropriate course of treatment to a patient.

SUMMARY OF THE INVENTION

The present invention provides methods and reagents for the rapiddetection of Neisseria gonorrhoeae that are species specific, that is,without substantial detection of other species in the Neisseria genus orspecies from other genera. For example, the nucleic acid detectionreagents of the invention (e.g., probe nucleic acids, sequence specificantibodies, etc.) typically bind to nucleotide sequences present in N.gonorrhoeae but not in other species. Further, since patients infectedwith N. gonorrhoeae are often also infected with Chlamydia trachomatis,the invention also provides methods of concurrently detecting N.gonorrhoeae and C. trachomatis in samples. This approach minimizes thenumber of diagnostic procedures to which a patient is subjected, whichalso typically minimizes the overall cost of diagnosis. In addition tocompositions and reaction mixtures, the invention also relates to kitsand systems for detecting these pathogenic agents, and to relatedcomputer and computer readable media.

In one aspect, the invention provides an oligonucleotide consisting of anucleic acid with a sequence selected from the group consisting of: SEQID NOS: 3-27, 37-60 or complements thereof. In another aspect, theinvention provides an oligonucleotide comprising a nucleic acid with asequence selected from the group consisting of: SEQ ID NOS: 3-27, 37-60and complements thereof, which oligonucleotide has 100 or fewernucleotides. In still another aspect, the invention provides anoligonucleotide that includes a nucleic acid having at least 90%sequence identity (e.g., at least 95%, etc.) to one of SEQ ID NOS: 3-27,37-60 or a complement thereof, which oligonucleotide has 100 or fewernucleotides. Typically, these oligonucleotides are primer nucleic acids,probe nucleic acids, or the like in these embodiments. In certain ofthese embodiments, the oligonucleotides have 40 or fewer nucleotides(e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, etc.). In someembodiments, the oligonucleotides comprise at least one modifiednucleotide. Optionally, the oligonucleotides comprise at least one labeland/or at least one quencher moiety. In some embodiments, theoligonucleotides include at least one conservatively modified variation.

In another aspect, the invention relates to an oligonucleotidecomprising at least 90% sequence identity with a subsequence of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the complement thereof, whicholigonucleotide has 100 or fewer nucleotides. In certain embodiments,the oligonucleotide has a sequence between about 12 and about 50nucleotides in length. In some embodiments, at least one nucleotide ofthe oligonucleotide is modified to alter nucleic acid hybridizationstability relative to unmodified nucleotides. In certain embodiments,the oligonucleotide comprises at least one label and/or at least onequencher moiety. In some embodiments, a solid support comprises theoligonucleotide.

In another aspect, the invention provides a method of detectingNeisseria gonorrhoeae in a sample, which method includes (a) contactingnucleic acids from the sample with at least a first pair of primernucleic acids that selectively bind to a nucleic acid with a sequenceconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36 or the variant, in at least onenucleic acid amplification reaction. The method also includes (b)detecting the nucleic acids and/or one or more amplicons thereof fromthe nucleic acid amplification reaction during or after (a), therebydetecting the Neisseria gonorrhoeae in the sample. In certainembodiments, for example, the nucleic acids and/or the amplicons thereofcomprise at least one sequence selected from the group consisting of:SEQ ID NOS: 28-33. In some embodiments, (a) comprises contacting thenucleic acids from the sample with at least a second pair of primernucleic acids that are at least partially complementary to a Chlamydiatrachomatis nucleic acid. In these embodiments, (b) comprises detectingone or more additional amplicons from the nucleic acid amplificationreaction during or after (a), thereby detecting Chlamydia trachomatis inthe sample. In certain embodiments, at least one of the primer nucleicacids comprises a modified primer nucleic acid. In some embodiments, atleast one of the primer nucleic acids comprises at least one label. Inthese embodiments, (b) optionally comprises detecting a detectablesignal produced by the label, or amplifying a detectable signal producedby the label to produce an amplified signal and detecting the amplifiedsignal. In some embodiments, (b) comprises monitoring binding betweenthe amplicons and one or more nucleic acid detection reagents thatdetectably bind to a nucleic acid with a sequence consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36 or the variant. Typically, at least one of the nucleicacid detection reagents comprises at least one label and/or at least onequencher moiety. In these embodiments, (b) optionally comprisesdetecting a detectable signal produced by the label, or amplifying adetectable signal produced by the label to produce an amplified signaland detecting the amplified signal.

In another aspect, the invention provides a method of determining apresence of Neisseria gonorrhoeae in a sample, which method comprises(a) contacting nucleic acids and/or amplicons thereof from the samplewith one or more oligonucleotides that selectively bind to a nucleicacid with a sequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, a substantially identical variant thereof in which the varianthas at least 90% sequence identity to one of SEQ ID NOS: 1 or 2 or 36,or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36 or thevariant. The method also includes (b) monitoring (e.g., at a single timepoint, at multiple discrete time points, continuously over a selectedtime period, etc.) binding between the nucleic acids and/or ampliconsthereof, and the oligonucleotides, in which detectable binding betweenthe nucleic acids and/or amplicons thereof, and the oligonucleotides,determines the presence of Neisseria gonorrhoeae in the sample. In someembodiments, for example, the nucleic acids and/or the amplicons thereofcomprise at least one sequence selected from the group consisting of:SEQ ID NOS: 28-33. The presence of Neisseria gonorrhoeae in the sampleis generally unknown or unsubstantiated before (a). In certainembodiments, (a) comprises contacting the nucleic acids and/or ampliconsthereof with the oligonucleotides in solution at a temperature of atleast 42° C. for at least 15 minutes in which a total weight of thesolution comprises about 50% formalin and comprises heparin at aconcentration of about 1 mg/ml. Moreover, the method typically comprisesa reaction other than a sequencing reaction. The sample is generallyderived from a mammalian subject, such as a human subject. In certainembodiments, the nucleic acids and/or amplicons thereof and theoligonucleotides are contacted in solution. Optionally, a solid supportcomprises the nucleic acids and/or amplicons (e.g., arrayed on the solidsupport). As an additional option, a solid support comprises theoligonucleotides.

In certain embodiments of the invention, the method further includescontacting the nucleic acids and/or amplicons thereof from the samplewith at least one additional oligonucleotide that detectably binds to aChlamydia trachomatis nucleic acid. In these embodiments, the methodalso includes monitoring the binding between the nucleic acids and/oramplicons thereof and the additional oligonucleotide, thereby detectingChlamydia trachomatis in the sample. In some embodiments, the methodincludes repeating (a) and (b) at least once using at least oneadditional sample (e.g., from the same subject) and comparing thebinding between the nucleic acids and/or amplicons thereof, and theoligonucleotides, of (b) with at least one repeated (b) to monitor,e.g., the course of treatment for a subject diagnosed with a Neisseriagonorrhoeae and/or a Chlamydia trachomatis infection, the recurrence ofthe infection, or the like.

The nucleic acid detection reagents of the invention include variousembodiments. To illustrate, at least one of the nucleic acid detectionreagents may comprise an oligonucleotide (e.g., a probe nucleic acid, aprimer nucleic acid, etc.). Typically, the oligonucleotide comprises atleast 85% (e.g., about 90%, about 95%, etc.) sequence identity with asubsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 1 or 2 or 36, or acomplement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36 or the variant.In some of these embodiments, (b) comprises monitoring binding betweenthe oligonucleotide and the nucleic acid and/or amplicons thereof.Optionally, the oligonucleotide has a sequence between about 8 and about100 nucleotides in length. In certain embodiments, the oligonucleotidehas a sequence selected from the group consisting of: SEQ ID NOS: 3-27and 37-60, a substantially identical variant thereof in which thevariant has at least 90% sequence identity to one of SEQ ID NOS: 3-27and 37-60, and complements of SEQ ID NOS: 3-27 and 37-60 and thevariant. Optionally, at least one nucleotide of the oligonucleotide ismodified. In some embodiments, for example, the nucleotide is modifiedto alter nucleic acid hybridization stability relative to unmodifiednucleotides.

To further illustrate, at least one of the nucleic acid detectionreagents optionally detectably binds to a nucleic acid segment thatcomprises one or more nucleotide positions of SEQ ID NO: 1 selected fromthe group consisting of: 259, 260, 262, 264, 265, 266, 268, 269, 273,275, 276, 277, 279, 297, 298, 300, 301, 302, 303, 304, 305, 306, 308,313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432,433, 434, 435, 440, 441, and 447. As an additional option, at least oneof the nucleic acid detection reagents detectably binds to a nucleicacid segment that comprises one or more nucleotide positions of SEQ IDNO: 2 selected from the group consisting of: 89, 90, 91, 92, 95, 98,101, 105, 106, 107, 216, 217, 220, 222, 223, 225, 233, 235, 236, 238,335, 336, 337, 338, 339, 342, 345, 346, and 351. In other embodiments,at least one of the nucleic acid detection reagents comprises, e.g., asequence specific antibody.

In certain embodiments, the nucleic acids, the amplicons thereof, and/orthe nucleic acid detection reagents comprise at least one label and/orat least one quencher moiety. For example, the label optionallycomprises a fluorescent dye, a weakly fluorescent label, anon-fluorescent label, a colorimetric label, a chemiluminescent label, abioluminescent label, an antibody, an antigen, biotin, a hapten, amass-modifying group, a radioisotope, an enzyme, or the like. In theseembodiments, (b) typically comprises detecting a detectable signalproduced by the label. To illustrate, (b) optionally comprises (i)amplifying a detectable signal produced by the label to produce anamplified signal, and (ii) detecting the amplified signal.

In some embodiments, at least one segment of the nucleic acids isamplified prior to or during (a) using at least one nucleic acidamplification technique to produce the amplicons and (b) comprisesmonitoring the binding between the nucleic acids and/or ampliconsthereof, and the nucleic acid detection reagents, during or afteramplification. For example, the nucleic acid amplification techniquetypically comprises a polymerase chain reaction, a ligase chainreaction, and/or the like. In these embodiments, the segment isoptionally amplified using at least one primer nucleic acid comprising asequence selected from the group consisting of: SEQ ID NOS: 3-27 and37-60, a substantially identical variant thereof in which the varianthas at least 90% sequence identity to one of SEQ ID NOS: 3-27 and 37-60,and complements of SEQ ID NOS: 3-27 and 37-60 and the variant. In someof these embodiments, the primer nucleic acid comprises at least onelabel, as described herein or otherwise known in the art. Optionally,the primer nucleic acid comprises a modified primer nucleic acid (e.g.,a nucleic acid amplification specificity altering modification, arestriction site linker, and/or the like).

In another aspect, the invention relates to a method of detectingNeisseria gonorrhoeae in a sample. The method includes (a) contactingnucleic acids from the sample with at least a first pair of primernucleic acids comprising at least one nucleic acid selected from thegroup consisting of: SEQ ID NOS: 3-27 and 37-60, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 3-27 and 37-60, and complements of SEQ IDNOS: 3-27 and 37-60 and the variant, in at least one nucleic acidamplification reaction. In addition, the method also includes (b)detecting the nucleic acids and/or one or more amplicons thereof fromthe nucleic acid amplification reaction during or after (a), therebydetecting the Neisseria gonorrhoeae in the sample. In certainembodiments, for example, the nucleic acids and/or the amplicons thereofcomprise at least one sequence selected from the group consisting of:SEQ ID NOS: 28-33. The sample is typically derived from a mammaliansubject, such as a human subject. Optionally, at least one of the primernucleic acids comprises a modified primer nucleic acid. In someembodiments, for example, the modified primer nucleic acid comprises anucleic acid amplification specificity altering modification and/or arestriction site linker modification. In certain embodiments, (a)comprises contacting the nucleic acids from the sample with at least asecond pair of primer nucleic acids that are at least partiallycomplementary to a Chlamydia trachomatis nucleic acid and (b) comprisesdetecting one or more additional amplicons from the nucleic acidamplification reaction during or after (a), thereby detecting Chlamydiatrachomatis in the sample.

In some embodiments, at least one of the primer nucleic acids comprisesat least one label. The label optionally comprises, e.g., a fluorescentdye, a weakly fluorescent label, a non-fluorescent label, a colorimetriclabel, a chemiluminescent label, a bioluminescent label, an antibody, anantigen, biotin, a hapten, a mass-modifying group, a radioisotope, anenzyme, etc. In these embodiments, (b) typically comprises detecting adetectable signal produced by the label. Optionally, (b) comprises (i)amplifying a detectable signal produced by the label to produce anamplified signal, and (ii) detecting the amplified signal.

In certain embodiments, (b) comprises monitoring binding between theamplicons and one or more nucleic acid detection reagents thatspecifically bind to a nucleic acid with a sequence consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36, or the variant. Optionally, at least one of thenucleic acid detection reagents comprises an oligonucleotide (e.g., aprobe nucleic acid, etc.). In some of these embodiments, (b) comprisesdetecting hybridization between the oligonucleotide and the amplicons.Optionally, the oligonucleotide comprises a sequence between about 8 andabout 100 nucleotides in length. In certain embodiments, at least onenucleotide of the oligonucleotide is modified (e.g., to alter nucleicacid hybridization stability relative to unmodified nucleotides or thelike). For example, at least one of the nucleic acid detection reagentscomprises a nucleic acid comprising a sequence selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60, a substantially identicalvariant thereof in which the variant has at least 90% sequence identityto one of SEQ ID NOS: 3-27 and 37-60, and complements of SEQ ID NOS:3-27 and 37-60 and the variant. To further illustrate, at least one ofthe nucleic acid detection reagents optionally detectably binds to anucleic acid segment that comprises one or more nucleotide positions ofSEQ ID NO: 1 selected from the group consisting of: 259, 260, 262, 264,265, 266, 268, 269, 273, 275, 276, 277, 279, 297, 298, 300, 301, 302,303, 304, 305, 306, 308, 313, 314, 315, 316, 317, 318, 320, 321, 325,326, 428, 429, 431, 432, 433, 434, 435, 440, 441, and 447. As anadditional option, at least one of the nucleic acid detection reagentsdetectably binds to a nucleic acid segment that comprises one or morenucleotide positions of SEQ ID NO: 2 selected from the group consistingof: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223,225, 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and351. In some embodiments, at least one of the nucleic acid detectionreagents comprises a sequence specific antibody or the like. Optionally,at least one of the nucleic acid detection reagents comprises at leastone label and/or at least one quencher moiety. An exemplary labeloptionally comprises a fluorescent dye, a weakly fluorescent label, anon-fluorescent label, a colorimetric label, a chemiluminescent label, abioluminescent label, an antibody, an antigen, biotin, a hapten, amass-modifying group, a radioisotope, an enzyme, or the like. In theseembodiments, (b) typically comprises detecting a detectable signalproduced by the label. In some of these embodiments, (b) comprises (i)amplifying a detectable signal produced by the label to produce anamplified signal, and (ii) detecting the amplified signal.

In another aspect, the invention provides a method of detectingNeisseria gonorrhoeae in a sample in which the method includes (a)contacting nucleic acids from the sample with at least a first pair ofprimer nucleic acids in at least one nucleic acid amplificationreaction, in which each of the primer nucleic acids have between 12 and100 nucleotides, and in which at least one of the primer nucleic acidscomprises at least 90% sequence identity with a subsequence of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or a complement thereof. The methodalso includes (b) detecting the nucleic acids and/or one or moreamplicons thereof from the nucleic acid amplification reaction during orafter (a), thereby detecting the Neisseria gonorrhoeae in the sample.Typically, the presence of Neisseria gonorrhoeae in the sample isunknown or unsubstantiated before (a). In some embodiments, one or moreof the primer nucleic acids has a sequence selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60, a substantially identicalvariant thereof wherein the variant has at least 90% sequence identityto one of SEQ ID NOS: 3-27 and 37-60, and complements of SEQ ID NOS:3-27 and 37-60 and the variant.

In still another aspect, the invention relates to a method ofdetermining a presence of Neisseria gonorrhoeae in a sample in which themethod includes (a) contacting nucleic acids and/or amplicons thereoffrom the sample with at least one oligonucleotide that has between 12and 100 nucleotides, which oligonucleotide comprises at least 90%sequence identity with a subsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 36, or a complement thereof. In addition, the method alsoincludes (b) monitoring binding between the nucleic acids and/oramplicons thereof, and the oligonucleotide, wherein detectable bindingbetween the nucleic acids and/or amplicons thereof, and theoligonucleotide, determines the presence of Neisseria gonorrhoeae in thesample. Typically, the presence of Neisseria gonorrhoeae in the sampleis unknown or unsubstantiated before (a). In certain embodiments, one ormore of the primer nucleic acids has a sequence selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60, a substantially identicalvariant thereof wherein the variant has at least 90% sequence identityto one of SEQ ID NOS: 3-27 and 37-60, and complements of SEQ ID NOS:3-27 and 37-60 and the variant.

In another aspect, the invention relates to a composition comprising asample derived from a subject and one or more nucleic acid detectionreagents that selectively bind to a nucleic acid with a sequenceconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant. A presence ofNeisseria gonorrhoeae in the sample is generally unknown orunsubstantiated. Typically, the nucleic acid detection reagents compriseat least one chemically synthesized nucleic acid. In certainembodiments, at least one of the nucleic acid detection reagentscomprises an oligonucleotide (e.g., a probe nucleic, a primer nucleicacid, or the like). Typically, the oligonucleotide comprises at least85% (e.g., about 90%, about 95%, etc.) sequence identity with asubsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or thecomplement thereof. In some of these embodiments, the oligonucleotidehas a sequence between about 8 and about 100 nucleotides in length(e.g., between about 12 and about 50 nucleotides in length). In certainembodiments, at least one nucleotide of the oligonucleotide is modified(e.g., to alter nucleic acid hybridization stability relative tounmodified nucleotides). For example, the nucleic acid detectionreagents optionally comprise at least one nucleic acid having a sequenceselected from the group consisting of: SEQ ID NOS: 3-27 and 37-60, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 3-27 and 37-60, andcomplements of SEQ ID NOS: 3-27 and 37-60 and the variant. To furtherillustrate, at least one of the nucleic acid detection reagentsoptionally detectably binds to a nucleic acid segment that comprises oneor more nucleotide positions of SEQ ID NO: 1 selected from the groupconsisting of: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276,277, 279, 297, 298, 300, 301, 302, 303, 304, 305, 306, 308, 313, 314,315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434,435, 440, 441, and 447. As an additional option, at least one of thenucleic acid detection reagents detectably binds to a nucleic acidsegment that comprises one or more nucleotide positions of SEQ ID NO: 2selected from the group consisting of: 89, 90, 91, 92, 95, 98, 101, 105,106, 107, 216, 217, 220, 222, 223, 225, 233, 235, 236, 238, 335, 336,337, 338, 339, 342, 345, 346, and 351. In some embodiments, the nucleicacid detection reagents comprise at least one sequence specificantibody. In certain embodiments, the composition further includes atleast one additional nucleic acid detection reagent that detectablybinds to a Chlamydia trachomatis nucleic acid.

Typically, at least one of the nucleic acid detection reagents comprisesat least one label and/or at least one quencher moiety. To illustrate,the label optionally comprises a fluorescent dye, a weakly fluorescentlabel, a non-fluorescent label, a colorimetric label, a chemiluminescentlabel, a bioluminescent label, an antibody, an antigen, biotin, ahapten, a mass-modifying group, a radioisotope, an enzyme, or the like.

The nucleic acid detection reagents of the compositions of the inventionare provided in various formats. In some embodiments, for example, atleast one of the nucleic acid detection reagents is in solution. Inother embodiments, a solid support comprises at least one of the nucleicacid detection reagents. In these embodiments, the nucleic aciddetection reagents are non-covalently or covalently attached to thesolid support. Exemplary solid supports utilized in these embodimentsare optionally selected from, e.g., a plate, a microwell plate, a bead,a microbead (e.g., a magnetic microbead, etc), a tube (e.g., amicrotube, etc.), a fiber, a whisker, a comb, a hybridization chip, amembrane, a single crystal, a ceramic layer, a self-assemblingmonolayer, and the like.

To further illustrate, the nucleic acid detection reagents areoptionally conjugated with biotin or a biotin derivative and the solidsupport is optionally conjugated with avidin or an avidin derivative, orstreptavidin or a streptavidin derivative. In some embodiments, a linkerattaches the nucleic acid detection reagents to the solid support. Thelinker is typically selected from, e.g., an oligopeptide, anoligonucleotide, an oligopolyamide, an oligoethyleneglycerol, anoligoacrylamide, an alkyl chain, and the like. Optionally, a cleavableattachment attaches the nucleic acid detection reagents to the solidsupport. The cleavable attachment is generally cleavable by, e.g., heat,an enzyme, a chemical agent, electromagnetic radiation, etc.

In other aspects, the invention provides a reaction mixture thatincludes a set of amplicons having sequences that correspond tosubsequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 1 or 2 or 36, or acomplement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant,which amplicons lack terminator nucleotides. Typically, at least asubset of the set of amplicons is produced using at least one primernucleic acid having a sequence selected from the group consisting of:SEQ ID NOS: 3-27 and 37-60, a substantially identical variant thereof inwhich the variant has at least 90% sequence identity to one of SEQ IDNOS: 3-27 and 37-60, and complements of SEQ ID NOS: 3-27 and 37-60 andthe variant. In certain embodiments, the primer nucleic acid comprises amodified primer nucleic acid. For example, the modified primer nucleicacid optionally comprises a nucleic acid amplification specificityaltering modification, a restriction site linker modification, and/orthe like. In some embodiments, the reaction mixture further includes anadditional set of amplicons that comprise sequences that correspond to aChlamydia trachomatis nucleic acid sequence.

In another aspect, the invention provides a kit that includes (a) atleast one oligonucleotide that has between 12 and 100 or fewnucleotides, which oligonucleotide comprises at least 90% sequenceidentity with a subsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:36, or a complement thereof; and one or more of: (b) instructions fordetermining a presence of Neisseria gonorrhoeae in a sample bymonitoring binding between nucleic acids and/or amplicons thereof fromthe sample and the oligonucleotide in which the presence of Neisseriagonorrhoeae in the sample is unknown or unsubstantiated, or (c) at leastone container for packaging at least the oligonucleotide. In some ofthese embodiments, the oligonucleotide has a sequence between about 8and about 100 nucleotides in length. In certain embodiments, forexample, the oligonucleotide has a sequence selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60, a substantially identicalvariant thereof in which the variant has at least 90% sequence identityto one of SEQ ID NOS: 3-27 and 37-60, and complements of SEQ ID NOS:3-27 and 37-60 and the variant. To further illustrate, theoligonucleotide optionally detectably binds to a nucleic acid segmentthat comprises one or more nucleotide positions of SEQ ID NO: 1 selectedfrom the group consisting of: 259, 260, 262, 264, 265, 266, 268, 269,273, 275, 276, 277, 279, 297, 298, 300, 301, 302, 303, 304, 305, 306,308, 313, 314, 315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431,432, 433, 434, 435, 440, 441, and 447. As an additional option, theoligonucleotide detectably binds to a nucleic acid segment thatcomprises one or more nucleotide positions of SEQ ID NO: 2 selected fromthe group consisting of: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107,216, 217, 220, 222, 223, 225, 233, 235, 236, 238, 335, 336, 337, 338,339, 342, 345, 346, and 351. In other embodiments, the nucleic aciddetection reagent is a sequence specific antibody. In certainembodiments, the kit further includes one or more nucleic acid detectionreagents that specifically bind to a Chlamydia trachomatis nucleic acid.In these embodiments, the kit typically further includes instructionsfor detecting Chlamydia trachomatis in the sample by monitoring bindingbetween nucleic acids and/or amplicons thereof from the sample and theadditional nucleic acid detection reagents, and/or one or morecontainers for packaging the additional nucleic acid detection reagents.In some embodiments, kit typically further includes at least one enzyme(e.g., a polymerase, etc.) and/or one or more nucleotides.

In some embodiments, the nucleic acid detection reagent is in solution,whereas in others, a solid support comprises the nucleic acid detectionreagent. The solid support is optionally selected from, e.g., a plate, amicrowell plate, a bead, a microbead, a tube, a fiber, a whisker, acomb, a hybridization chip, a membrane, a single crystal, a ceramiclayer, a self-assembling monolayer, or the like.

Typically, the oligonucleotide comprises at least one label and/or atleast one quencher moiety. Exemplary labels include, e.g., a fluorescentdye, a weakly fluorescent label, a non-fluorescent label, a colorimetriclabel, a chemiluminescent label, a bioluminescent label, an antibody, anantigen, biotin, a hapten, a mass-modifying group, a radioisotope, anenzyme, or the like.

In still other aspects, the invention provides a system (e.g., anautomated system) for detecting Neisseria gonorrhoeae in a sample. Thesystem includes (a) at least one oligonucleotide that has between 12 and100 or few nucleotides, which oligonucleotide comprises at least 90%sequence identity with a subsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 36, or a complement thereof. The system also includes (b) atleast one detector that detects binding between nucleic acids and/oramplicons thereof from the sample and the oligonucleotide, and (c) atleast one controller operably connected to the detector, whichcontroller comprises one or more instructions sets that correlate thebinding detected by the detector with a presence of Neisseriagonorrhoeae in the sample. The oligonucleotide typically has a sequenceselected from the group consisting of: SEQ ID NOS: 3-27 and 37-60 orcomplements thereof. To further illustrate, the oligonucleotideoptionally detectably binds to a nucleic acid segment that comprises oneor more nucleotide positions of SEQ ID NO: 1 selected from the groupconsisting of: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276,277, 279, 297, 298, 300, 301, 302, 303, 304, 305, 306, 308, 313, 314,315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434,435, 440, 441, and 447. As an additional option, the oligonucleotidedetectably binds to a nucleic acid segment that comprises one or morenucleotide positions of SEQ ID NO: 2 selected from the group consistingof: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217, 220, 222, 223,225, 233, 235, 236, 238, 335, 336, 337, 338, 339, 342, 345, 346, and351. In addition, the oligonucleotide typically comprises at least onelabel and/or at least one quencher moiety. In certain embodiments, thesystem further includes one or more additional nucleic acid detectionreagents that specifically bind to a Chlamydia trachomatis nucleic acidin which the detector detects binding between the nucleic acids and/oramplicons thereof from the sample and the additional nucleic aciddetection reagents, and in which the controller comprises at least oneinstruction set that correlates the binding detected by the detectorwith a presence of Chlamydia trachomatis in the sample. In someembodiments, at least one container or solid support comprises theoligonucleotide. In these embodiments, the system optionally furtherincludes (d) at least one thermal modulator operably connected to thecontainer or solid support to modulate temperature in the container oron the solid support, and/or (e) at least one fluid transfer componentthat transfers fluid to and/or from the container or solid support,e.g., for performing one or more nucleic acid amplification techniquesin the container or on the solid support, etc.

In another aspect, the invention provides a system that includes (a)computer or computer readable medium comprising a data set thatcomprises a plurality of character strings that correspond to aplurality of sequences that correspond to subsequences of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variant thereofin which the variant has at least 90% sequence identity to one of SEQ IDNOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, or the variant. The system also includes (b) an automaticsynthesizer coupled to an output of the computer or computer readablemedium, which automatic synthesizer accepts instructions from thecomputer or computer readable medium, which instructions directsynthesis of one or more nucleic acids that correspond to one or morecharacter strings in the data set. Typically, at least one of thecharacter strings corresponds to a sequence selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60 or complements thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of a Neisseria gonorrhoeae DirectRepeat 9 (NGDR9) sequence (SEQ ID NO: 1) with the sequences of ampliconsof genomic DNA from various N. gonorrhoeae strains (strain 1117, SEQ IDNO:29; strain 11205 SEQ ID NO:30; strain 6346, SEQ ID NO:31; strain6359, SEQ ID NO:32; and strain 6364, SEQ ID NO:33). The majority(consensus) sequence is SEQ ID NO:28.

FIG. 2 is a block diagram showing a representative example system fordetecting N. gonorrhoeae in a sample.

FIG. 3 is a block diagram showing a representative example systemincluding a computer and a computer readable medium in which variousaspects of the present invention may be embodied.

FIG. 4 shows a ClustalW alignment of the NGDR9 sequence (SEQ ID NO: 1)with a portion (SEQ ID NO:34) of the sequence of Brucella suis 1330chromosome I section 155 (GenBank® accession number AE014469).

FIGS. 5 A and B are photographs of agarose gels that show the detectionof a 190 base pair segment of NGDR9.

FIGS. 6 A and B are photographs of agarose gels that show the detectionof a 190 base pair segment of NGDR9.

FIG. 7 is a photograph of an agarose gel that shows the detection of a416 base pair segment of NGDR9.

FIG. 8 depicts a ClustalW alignment of the Neisseria gonorrhoeae DirectRepeat 33 (NGDR33) (SEQ ID NO:2) sequence with a portion (SEQ ID NO:35)of the sequence of Neisseria meningitidis serogroup B strain MC58section 77 (GenBank® accession number AE002435).

FIGS. 9 A and B are photographs of agarose gels that show the detectionof a 265 base pair segment of NGDR33.

FIG. 10 is a photograph of an agarose gel that shows the detection of a265 base pair segment of NGDR33.

FIG. 11 shows a sequence alignment of a Neisseria gonorrhoeae DirectRepeat 9 Variant (NGDR9Var) sequence (SEQ ID NO: 36) with the sequencesof amplicons of genomic DNA from various N. gonorrhoeae strains (strain1137, SEQ ID NO: 61; strain 6676, SEQ ID NO: 62; strain 6677, SEQ ID NO:63; strain 6864A, SEQ ID NO: 64; strain 2072, SEQ ID NO: 65; strain3533, SEQ ID NO: 66; and strain 6864B, SEQ ID NO: 67) and the NGDR9sequence (SEQ ID NO: 1).

FIGS. 12A and 12B are photographs of agarose gels that show thedetection of a 215 base pair segment of NGDR9Var.

FIGS. 13A and 13B are photographs of agarose gels that show thesimultaneous detection of a 473 base pair segment of NGDR9 and a 394base pair segment of NGDR9Var.

DETAILED DESCRIPTION I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularoligonucleotide probes, methods, compositions, reaction mixtures, kits,systems, computers, or computer readable media, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. Further, unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention pertains. In describing and claiming the present invention,the following terminology and grammatical variants will be used inaccordance with the definitions set forth below.

A “5′-nuclease probe” refers to an oligonucleotide probe that comprisesat least two labels and emits radiation of increased intensity after oneof the two labels is cleaved or otherwise separated from the probe. Incertain embodiments, for example, a 5′-nuclease probe is labeled withtwo different fluorescent dyes, e.g., a 5′ terminus reporter dye and the3′ terminus quenching dye or moiety. When the probe is intact, energytransfer typically occurs between the two fluorophores such thatfluorescent emission from the reporter dye is quenched. During anextension step of a polymerase chain reaction, for example, a5′-nuclease probe bound to a template nucleic acid is cleaved by the 5′nuclease activity of, e.g., a Taq polymerase such that the fluorescentemission of the reporter dye is no longer quenched. Exemplary5′-nuclease probes are described in, e.g., U.S. Pat. No. 5,210,015,entitled “HOMOGENEOUS ASSAY SYSTEM USING THE NUCLEASE ACTIVITY OF ANUCLEIC ACID POLYMERASE,” issued May 11, 1993 to Gelfand et al., U.S.Pat. No. 5,994,056, entitled “HOMOGENEOUS METHODS FOR NUCLEIC ACIDAMPLIFICATION AND DETECTION,” issued Nov. 30, 1999 to Higuchi, and U.S.Pat. No. 6,171,785, entitled “METHODS AND DEVICES FOR HEMOGENEOUSNUCLEIC ACID AMPLIFICATION AND DETECTOR,” issued Jan. 9, 2001 toHiguchi, which are each incorporated by reference.

The term “alteration” refers to a change in a nucleic acid sequence,including, but not limited to, a substitution, an insertion, and/or adeletion.

An “amplification reaction” refers to a primer initiated replication ofone or more target nucleic acid sequences or complements thereto.

An “amplicon” refers to a molecule made by copying or transcribinganother molecule, e.g., as occurs in transcription, cloning, and/or in apolymerase chain reaction (“PCR”) (e.g., strand displacement PCRamplification (SDA), duplex PCR amplification, etc.) or other nucleicacid amplification technique. Typically, an amplicon is a copy of aselected nucleic acid (e.g., a template or target nucleic acid) or iscomplementary thereto.

An “amplified signal” refers to increased detectable signal that can beproduced in the absence of, or in conjunction with, an amplificationreaction. Exemplary signal amplification techniques are described in,e.g., Cao et al. (1995) “Clinical evaluation of branched DNA signalamplification for quantifying HIV type 1 in human plasma,” AIDS Res HumRetroviruses 11(3):353-361, and in U.S. Pat. No. 5,437,977 to Segev,U.S. Pat. No. 6,033,853 to Delair et al., and U.S. Pat. No. 6,180,777 toHorn, which are each incorporated by reference.

“Antibody” refers to a polypeptide substantially encoded by at least oneimmunoglobulin gene or fragments of at least one immunoglobulin gene,that can participate in detectable binding with a ligand. The termincludes naturally-occurring forms, as well as fragments andderivatives. Fragments within the scope of the term as used hereininclude those produced by digestion with various peptidases, such asFab, Fab′ and F(ab)′2 fragments, those produced by chemicaldissociation, by chemical cleavage, so long as the fragment remainscapable of detectable binding to a target molecule, such as an antigenindicative of a disease.

An “array” refers to an assemblage of elements. The assemblage can bespatially ordered (a “patterned array”) or disordered (a “randomlypatterned” array). The array can form or comprise one or more functionalelements (e.g., a probe region on a microarray) or it can benon-functional.

The term “attached” or “conjugated” refers to interactions and/or statesin which material or compounds are connected or otherwise joined withone another. These interactions and/or states are typically produced by,e.g., covalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof. In certain embodiments, for example,oligonucleotide probes are attached to solid supports. In some of theseembodiments, an oligonucleotide probe is conjugated with biotin (i.e.,is biotinylated) and a solid support is conjugated with avidin such thatthe probe attaches to the solid support via the binding interaction of,e.g., biotin and avidin.

Molecular species “bind” when they associate with one another viacovalent and/or non-covalent interactions. For example, twocomplementary single-stranded nucleic acids can hybridize with oneanother to form a nucleic acid with at least one double-stranded region.To further illustrate, antibodies and corresponding antigens can alsonon-covalently associate with one another.

The term “cleavage” refers to a process of releasing a material orcompound from attachment to another material or compound. In certainembodiments, for example, oligonucleotides are cleaved from, e.g., asolid support to permit analysis of the oligonucleotides bysolution-phase methods. See, e.g., Wells et al. (1998) “Cleavage andAnalysis of Material from Single Resin Beads,” J. Org. Chem. 63:6430,which is incorporated by reference.

A “character” when used in reference to a character of a characterstring refers to a subunit of the string. In one embodiment, thecharacter of a character string encodes one subunit of an encodedbiological molecule. Thus, for example, where the encoded biologicalmolecule is a polynucleotide or oligonucleotide, a character of thestring encodes a single nucleotide.

A “character string” is any entity capable of storing sequenceinformation (e.g., the subunit structure of a biological molecule suchas the nucleotide sequence of a nucleic acid, etc.). In one embodiment,the character string can be a simple sequence of characters (letters,numbers, or other symbols) or it can be a numeric or codedrepresentation of such information in tangible or intangible (e.g.,electronic, magnetic, etc.) form. The character string need not be“linear,” but can also exist in a number of other forms, e.g., a linkedlist or other non-linear array (e.g., used as a code to generate alinear array of characters), or the like. Character strings aretypically those which encode oligonucleotide or polynucleotide strings,directly or indirectly, including any encrypted strings, or images, orarrangements of objects which can be transformed unambiguously tocharacter strings representing sequences of monomers or multimers inpolynucleotides, or the like (whether made of natural or artificialmonomers).

The term “Chlamydia trachomatis,” “C. trachomatis,” or “CT” refers thebacterial species trachomatis of the Chlamydia genus. See, e.g.,Stephens et al. (1998) “Genome sequence of an obligate intracellularpathogen of humans: Chlamydia trachomatis,” Science 282:754-759, Kalmanet al. (1999) “Comparative genomes of Chlamydia pneumoniae and C.trachomatis,” Nature Genetics 21:385-389, and Stephens, Chlamydia:Intracellular Biology, Pathogenesis, and Immunity, ASM Press (1999),which are each incorporated by reference. An exemplary GenBank®accession number for the complete sequence of the Chlamydia trachomatisgenome is NC_(—)000117. See also, the Chlamydia trachomatis database,which is on the world wide web at stdgen.lanl.gov as of Mar. 12, 2004.

The term “Chlamydia trachomatis nucleic acid” or “C. trachomatis nucleicacid” refers to a nucleic acid (and/or an amplicon thereof) that isderived or isolated from Chlamydia trachomatis.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

A “composition” refers to a combination of two or more differentcomponents. In certain embodiments, for example, a composition includesa solid support that comprises one or more oligonucleotide probes, e.g.,covalently or non-covalently attached to a surface of the support. Inother embodiments, a composition includes one or more oligonucleotideprobes in solution.

The term “deletion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is removed from thenucleic acid sequence, e.g., from a 5′-terminus, from a 3′-terminus,and/or from an internal position of the nucleic acid sequence.

The term “derivative” refers to a chemical substance relatedstructurally to another substance, or a chemical substance that can bemade from another substance (i.e., the substance it is derived from),e.g., through chemical or enzymatic modification. To illustrate,oligonucleotide probes are optionally conjugated with biotin or a biotinderivative. To further illustrate, one nucleic acid can be “derived”from another through processes, such as chemical synthesis based onknowledge of the sequence of the other nucleic acid, amplification ofthe other nucleic acid, or the like.

The term “detectably bind” refers to binding between at least twomolecular species (e.g., a probe nucleic acid and a target nucleic acid,a sequence specific antibody and a target nucleic acid, etc.) that isdetectable above a background signal (e.g., noise) using one or moremethods of detection.

Nucleic acids are “extended” or “elongated” when additional nucleotides(or other analogous molecules) are incorporated into the nucleic acids.For example, a nucleic acid is optionally extended by a nucleotideincorporating biocatalyst, such as a polymerase that typically addsnucleotides at the 3′ terminal end of a nucleic acid.

An “extended primer nucleic acid” refers to a primer nucleic acid towhich one or more additional nucleotides have been added or otherwiseincorporated (e.g., covalently bonded thereto).

Nucleic acids “hybridize” or “bind” when they associate with oneanother, typically in solution. Nucleic acids hybridize due to a varietyof well characterized physico-chemical forces, such as hydrogen bonding,solvent exclusion, base stacking and the like. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,New York), as well as in Ausubel (Ed.) Current Protocols in MolecularBiology, Volumes I, II, and III, 1997, which is incorporated byreference. Hames and Higgins (1995) Gene Probes 1 IRL Press at OxfordUniversity Press, Oxford, England, (Hames and Higgins 1) and flames andHiggins (1995) Gene Probes 2 IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides. Both Hames and Higgins 1 and 2 are incorporated byreference.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization assays or experiments, such as nucleic acid amplificationreactions, Southern and northern hybridizations, or the like, aresequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.

For purposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be at least about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids on a filter in a Southern or northern blotis 50% formalin with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of stringent wash conditions isa 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001), which is incorporated byreference, for a description of SSC buffer). Often the high stringencywash is preceded by a low stringency wash to remove background probesignal. An example low stringency wash is 2×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 5× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

Comparative hybridization can be used to identify nucleic acids of theinvention.

In particular, detection of stringent hybridization in the context ofthe present invention indicates strong structural similarity to, e.g.,the nucleic acids provided in the sequence listing herein. For example,it is desirable to identify test nucleic acids that hybridize to theexemplar nucleic acids herein under stringent conditions. One measure ofstringent hybridization is the ability to detectably hybridize to one ofthe listed nucleic acids (e.g., nucleic acids with sequences selectedfrom SEQ ID NOS: 3-27 and complements thereof) under stringentconditions. Stringent hybridization and wash conditions can easily bedetermined empirically for any test nucleic acid.

For example, in determining highly stringent hybridization and washconditions, the stringency of the hybridization and wash conditions aregradually increased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents such as formalin in the hybridizationor wash), until a selected set of criteria is met. For example, thestringency of the hybridization and wash conditions are graduallyincreased until a probe consisting of or comprising one or more nucleicacid sequences selected from SEQ ID NOS: 3-27 and 37-60 andcomplementary polynucleotide sequences thereof, binds to a perfectlymatched complementary target (again, a nucleic acid comprising one ormore nucleic acid sequences selected from SEQ ID NOS: 3-27 and 37-60 andcomplementary polynucleotide sequences thereof, with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe probe to a non-target nucleic acid. In this case, non-target nucleicacids are those from organisms other than N. gonorrhoeae and in certainembodiments, C. trachomatis. Examples of such non-target nucleic acidsinclude, e.g., those with GenBank® accession numbers, such as AE01469(Brucella suis 1330 chromosome I section 155) and AE002435 (Neisseriameningitidis serogroup B strain MC58 section 77). Additional suchsequences can be identified in, e.g., GenBank® by one of skill in theart.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least one-half as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least one-half as high as hybridization of the probe to thetarget under conditions in which the perfectly matched probe binds tothe perfectly matched complementary target with a signal to noise ratiothat is at least about 5×-10× as high as that observed for hybridizationto the non-target nucleic acids AE01469 (Brucella suis 1330 chromosome Isection 155) or AE002435 (Neisseria meningitidis serogroup B strain MC58section 77).

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to the non-target nucleic acidsAE01469 (Brucella suis 1330 chromosome I section 155) or AE002435(Neisseria meningitidis serogroup B strain MC58 section 77). A targetnucleic acid which hybridizes to a probe under such conditions, with asignal to noise ratio of at least one-half that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the stringency of hybridization and/or washconditions of the relevant hybridization assay. For example, those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×,20×, 50×, 100×, or 500× or more as high as that observed forhybridization to the non-target nucleic acids AE01469 (Brucella suis1330 chromosome I section 155) or AE002435 (Neisseria meningitidisserogroup B strain MC58 section 77) can be identified. A target nucleicacid which hybridizes to a probe under such conditions, with a signal tonoise ratio of at least one-half that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-ultra-high stringency conditions.

The detection of target nucleic acids which hybridize to the nucleicacids represented by SEQ ID NOS: 3-27 and 37-60 under high, ultra-highand ultra-ultra high stringency conditions are a feature of theinvention. Examples of such nucleic acids include those with one or afew silent or conservative nucleic acid substitutions as compared to agiven nucleic acid sequence.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, e.g., as measured usingone of the sequence comparison algorithms available to persons of skillor by visual inspection. Exemplary algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST programs, which are described in, e.g., Altschul et al. (1990)“Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish etal. (1993) “Identification of protein coding regions by databasesimilarity search” Nature Genet. 3:266-272, Madden et al. (1996)“Applications of network BLAST server” Meth. Enzymol. 266:131-141,Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs” Nucleic Acids Res. 25:3389-3402, andZhang et al. (1997) “PowerBLAST: A new network BLAST application forinteractive or automated sequence analysis and annotation” Genome Res.7:649-656, which are each incorporated by reference. Many other optimalalignment algorithms are also known in the art and are optionallyutilized to determine percent sequence identity.

The phrase “in solution” refers to an assay or reaction condition inwhich the components of the assay or reaction are not attached to asolid support and are present in a liquid medium. Exemplary liquidmediums include aqueous and organic fluids. For example, certain assaysof the invention include incubating oligonucleotide probes together withN. gonorrhoeae nucleic acids and N. gonorrhoeae nucleic acid ampliconsin solution to allow hybridization to occur.

The term “insertion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is added to the nucleicacid sequence, e.g., at a 5′-terminus, at a 3′-terminus, and/or at aninternal position of the nucleic acid sequence.

A “label” refers to a moiety attached (covalently or non-covalently), orcapable of being attached, to a molecule, which moiety provides or iscapable of providing information about the molecule (e.g., descriptive,identifying, etc. information about the molecule) or another moleculewith which the labeled molecule interacts (e.g., hybridizes, etc.).Exemplary labels include fluorescent labels (including, e.g., quenchersor absorbers), weakly fluorescent labels, non-fluorescent labels,colorimetric labels, chemiluminescent labels, bioluminescent labels,radioactive labels, mass-modifying groups, antibodies, antigens, biotin,haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), andthe like.

A “linker” refers to a chemical moiety that covalently or non-covalentlyattaches a compound or substituent group to another moiety, e.g., anucleic acid, an oligonucleotide probe, a primer nucleic acid, anamplicon, a solid support, or the like. For example, linkers areoptionally used to attach oligonucleotide probes to a solid support(e.g., in a linear or other logic probe array). To further illustrate, alinker optionally attaches a label (e.g., a fluorescent dye, aradioisotope, etc.) to an oligonucleotide probe, a primer nucleic acid,or the like. Linkers are typically at least bifunctional chemicalmoieties and in certain embodiments, they comprise cleavableattachments, which can be cleaved by, e.g., heat, an enzyme, a chemicalagent, electromagnetic radiation, etc. to release materials or compoundsfrom, e.g., a solid support. A careful choice of linker allows cleavageto be performed under appropriate conditions compatible with thestability of the compound and assay method. Generally a linker has nospecific biological activity other than to, e.g., join chemical speciestogether or to preserve some minimum distance or other spatialrelationship between such species. However, the constituents of a linkermay he selected to influence some property of the linked chemicalspecies such as three-dimensional conformation, net charge,hydrophobicity, etc. Exemplary linkers include, e.g., oligopeptides,oligonucleotides, oligopolyamides, oligoethyleneglycerols,oligoacrylamides, alkyl chains, or the like. Additional description oflinker molecules is provided in, e.g., Hermanson, BioconjugateTechniques, Elsevier Science (1996), Lyttle et al. (1996) Nucleic AcidsRes. 24(14):2793, Shchepino et al. (2001) Nucleosides, Nucleotides, &Nucleic Acids 20:369, Doronina et al (2001) Nucleosides, Nucleotides, &Nucleic Acids 20:1007, Trawick et al. (2001) Bioconjugate Chem. 12:900,Olejnik et al. (1998) Methods in Enzymology 291:135, and Pljevaljcic etal. (2003) J. Am. Chem. Soc. 125(12):3486, all of which are incorporatedby reference.

A “mass modifying” group modifies the mass, typically measured in termsof molecular weight as daltons, of a molecule that comprises the group.For example, mass modifying groups that increase the discriminationbetween at least two nucleic acids with single base differences in sizeor sequence can be used to facilitate sequencing using, e.g., molecularweight determinations.

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction. An “amplificationreaction mixture” refers to a solution containing reagents necessary tocarry out an amplification reaction, and typically contains primers, athermostable DNA polymerase, dNTP's, and a divalent metal cation in asuitable buffer. A reaction mixture is referred to as complete if itcontains all reagents necessary to carry out the reaction, andincomplete if it contains only a subset of the necessary reagents. Itwill be understood by one of skill in the art that reaction componentsare routinely stored as separate solutions, each containing a subset ofthe total components, for reasons of convenience, storage stability, orto allow for application-dependent adjustment of the componentconcentrations, and, that reaction components are combined prior to thereaction to create a complete reaction mixture. Furthermore, it will beunderstood by one of skill in the art that reaction components arepackaged separately for commercialization and that useful commercialkits may contain any subset of the reaction components, which includesthe modified primers of the invention.

A “modified primer nucleic acid” refers to a primer nucleic acid thatcomprises a moiety or sequence of nucleotides that provides a desiredproperty to the primer nucleic acid. In certain embodiments, forexample, modified primer nucleic acids comprise “nucleic acidamplification specificity altering modifications” that, e.g., reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like. Examples of nucleic acid amplificationspecificity altering modifications are described in, e.g., U.S. Pat. No.6,001,611, entitled “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS,”issued Dec. 14, 1999 to Will, which is incorporated by reference. Otherexemplary primer nucleic acid modifications include a “restriction sitelinker modification” in which a nucleotide sequence comprising aselected restriction site is attached, e.g., at 5′-terminus of a primernucleic acid. Restriction site linkers are typically attached to primernucleic acids to facilitate subsequent amplicon cloning or the like.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, an oligonucleotide probeoptionally comprises a quencher moiety, a labeling moiety, or the like.

The term “Neisseria gonorrhoeae,” “N. gonorrhoeae,” or “NG” refers tothe bacterial species gonorrhoeae of the Neisseria genus. See, e.g.,Schoolnik (Ed.) Pathogenic Neisseriae: Proceedings of the FourthInternational Symposium, Asilomar, California, 21-25 Oct. 1984, Amer.Society for Microbiology (1986), which is incorporated by reference.Additional general description of N. gonorrhoeae and C. trachomatis isprovided in, e.g., Struthers and Westran, Clinical Bacteriology, ASMPress and Manson Publishing (2003), Persing et al., MolecularMicrobiology: Diagnostic Principles and Practice, ASM Press (2003),Murray, Manual of Clinical Microbiology, 8th Ed., ASM Press (2003),which are each incorporated by reference. See also, the Neisseriagonorrhoeae database provided on the world wide web at stdgen.lanl.govas of Mar. 12, 2004.

The term “Neisseria gonorrhoeae nucleic acid” or “N. gonorrhoeae nucleicacid” refers to a nucleic acid (and/or an amplicon thereof) that isderived or isolated from Neisseria gonorrhoeae.

The term “nucleic acid” refers to nucleotides (e.g., ribonucleotides,deoxyribonucleotides, dideoxynucleotides, etc.) and polymers thatcomprise such nucleotides covalently linked together, either in a linearor branched fashion. Exemplary nucleic acids include deoxyribonucleoicacids (DNAs), ribonucleic acids (RNAs), DNA-RNA hybrids,oligonucleotides, polynucleotides, genes, cDNAs, aptamers, antisensenucleic acids, interfering RNAs (RNAis), molecular beacons, nucleic acidprobes, peptide nucleic acids (PNAs), locked nucleic acids (LNA™s),PNA-DNA conjugates, PNA-RNA conjugates, LNA™-DNA conjugates, LNA™-RNAconjugates, etc.

A nucleic acid is typically single-stranded or double-stranded and willgenerally contain phosphodiester bonds, although in some cases, asoutlined herein, nucleic acid analogs are included that may havealternate backbones, including, for example and without limitation,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925 andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al.(1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419, which are each incorporated by reference),phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S.Pat. No. 5,644,048, which are both incorporated by reference),phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, whichis incorporated by reference), O-methylphosphoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press (1992), which is incorporated by reference), andpeptide nucleic acid backbones and linkages (see, Egholm (1992) J. Am.Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31:1008;Nielsen (1993) Nature 365:566; and Carlsson et al. (1996) Nature380:207, which are each incorporated by reference). Other analog nucleicacids include those with positively charged backbones (Denpcy et al.(1995) Proc. Natl. Acad. Sci. USA 92:6097, which is incorporated byreference); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl. Ed. English30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsingeret al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; and Tetrahedron Lett. 37:743 (1996), which areeach incorporated by reference) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Ed. Y. S. Sanghvi and P. Dan Cook, which referencesare each incorporated by reference. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al. (1995) Chem. Soc. Rev. pp 169-176, which isincorporated by reference). Several nucleic acid analogs are alsodescribed in, e.g., Rawls, C & E News Jun. 2, 1997 page 35, which isincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to alter the stability and half-life of suchmolecules in physiological environments.

In addition to these naturally occurring heterocyclic bases that aretypically found in nucleic acids (e.g., adenine, guanine, thymine,cytosine, and uracil), nucleic acid analogs also include those havingnon-naturally occurring heterocyclic or modified bases, many of whichare described, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640,which are each incorporated by reference. To further illustrate, certainbases used in nucleotides that act as melting temperature (T_(m))modifiers are optionally included. For example, some of these include7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.),pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, entitled“SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINE NUCLEOTIDES,” which issued Nov.23, 1999 to Seela, which is incorporated by reference. Otherrepresentative heterocyclic bases include, e.g., hypoxanthine, inosine,xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine,2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine andxanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, and the like.

Examples of modified bases and nucleotides are also described in, e.g.,U.S. Pat. No. 5,484,908, entitled “OLIGONUCLEOTIDES CONTAINING5-PROPYNYL PYRIMIDINES,” issued Jan. 16, 1996 to Froehler et al., U.S.Pat. No. 5,645,985, entitled “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Jul.8, 1997 to Froehler et al., U.S. Pat. No. 5,830,653, entitled “METHODSOF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Nov. 3, 1998to Froehler et al., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al.,U.S. Pat. No. 6,303,315, entitled “ONE STEP SAMPLE PREPARATION ANDDETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct.16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905,entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al.that published May 15, 2003, which are each incorporated by reference.

The term “nucleic acid detection reagent” refers to a reagent thatdetectably binds (e.g., hydrogen bonds in nucleic acid hybridization, inantibody-antigen recognition, or the like, or other types of bindinginteractions) to a nucleic acid that comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36, a substantially identical variant thereof in which thevariant has at least 90% sequence identity to one of SEQ ID NOS: 1 or 2or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, orthe variant. For example, nucleic acids (e.g., probe nucleic acids,primer nucleic acids, etc.) that comprise sequences selected from SEQ IDNOS: 3-27 and 37-60 or complements thereof specifically bind to nucleicacids having these sequences. Other exemplary nucleic acid detectionreagents include sequence specific antibodies that specifically bind tonucleic acids comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 1 or 2 or 36, or acomplement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant.

A “nucleotide” refers to an ester of a nucleoside, e.g., a phosphateester of a nucleoside. For example, a nucleotide can include 1, 2, 3, ormore phosphate groups covalently linked to a 5′ position of a sugarmoiety of the nucleoside.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein- and/or nucleic acid-based catalyst that acts to reduce theactivation energy of a chemical reaction involving other compounds or“substrates.” A “nucleotide incorporating enzyme” refers to an enzymethat catalyzes the incorporation of nucleotides into a nucleic acid,e.g., during nucleic acid amplification or the like. Exemplarynucleotide incorporating enzymes include, e.g., polymerases, terminaltransferases, reverse transcriptases, telomerases, polynucleotidephosphorylases, and the like.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22:1859-1862; the triester method of Matteucci et al. (1981) J.Am. Chem. Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, or other methods known in theart. All of these references are incorporated by reference.

The term “oligonucleotide probe,” “probe nucleic acid,” or “probe”refers to a labeled or unlabeled oligonucleotide capable of selectivelyhybridizing to a target nucleic acid under suitable conditions.Typically, a probe is sufficiently complementary to a specific targetsequence (e.g., an N. gonorrhoeae nucleic acid that comprises SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36, or the variant), a C. trachomatis nucleic acidsequence, etc.) contained in a nucleic acid sample to form a stablehybridization duplex with the target sequence under a selectedhybridization condition, such as, but not limited to, a stringenthybridization condition. A hybridization assay carried out using theprobe under sufficiently stringent hybridization conditions permits theselective detection of a specific target sequence. The term “hybridizingregion” refers to that region of a nucleic acid that is exactly orsubstantially complementary to, and therefore hybridizes to, the targetsequence. For use in a hybridization assay for the discrimination ofsingle nucleotide differences in sequence, the hybridizing region istypically from about 8 to about 100 nucleotides in length. Although thehybridizing region generally refers to the entire oligonucleotide, theprobe may include additional nucleotide sequences that function, forexample, as linker binding sites to provide a site for attaching theprobe sequence to a solid support or the like. In certain embodiments,an oligonucleotide probe of the invention comprises one or more labels(e.g., a reporter dye, a quencher moiety, etc.), such as a FRET probe, amolecular beacon, or the like, which can also be utilized to detecthybridization between the probe and target nucleic acids in a sample. Insome embodiments, the hybridizing region of the oligonucleotide probe iscompletely complementary to the target sequence. However, in general,complete complementarity is not necessary; stable duplexes may containmismatched bases or unmatched bases. Modification of the stringentconditions may be necessary to permit a stable hybridization duplex withone or more base pair mismatches or unmatched bases. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001), which is incorporatedby reference, provides guidance for suitable modification. Stability ofthe target/probe duplex depends on a number of variables includinglength of the oligonucleotide, base composition and sequence of theoligonucleotide, temperature, and ionic conditions. One of skill in theart will recognize that, in general, the exact complement of a givenprobe is similarly useful as a probe. Exemplary probes of the invention,which bind to an N. gonorrhoeae nucleic acid with a sequence consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identicalvariant thereof in which the variant has at least 90% sequence identityto one of SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 36, or the variant, comprise sequences selectedfrom SEQ ID NOS: 3-27 and 37-60 and complements thereof. One of skill inthe art will also recognize that, in certain embodiments, probe nucleicacids can also be used as primer nucleic acids.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a template nucleic acid (e.g., an N. gonorrhoeae nucleic acid thatcomprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant, a C. trachomatisnucleic acid, etc.) and permit chain extension or elongation using,e.g., a nucleotide incorporating biocatalyst, such as a polymerase underappropriate reaction conditions. A primer nucleic acid is typically anatural or synthetic oligonucleotide (e.g., a single-strandedoligodeoxyribonucleotide, etc.). Although other primer nucleic acidlengths are optionally utilized, they typically comprise hybridizingregions that range from about 8 to about 100 nucleotides in length.Short primer nucleic acids generally utilize cooler temperatures to formsufficiently stable hybrid complexes with template N. gonorrhoeae or C.trachomatis nucleic acid. A primer nucleic acid that is at leastpartially complementary to a subsequence of a template N. gonorrhoeae orC. trachomatis nucleic acid is typically sufficient to hybridize withthe template for extension to occur. A primer nucleic acid can belabeled, if desired, by incorporating a label detectable by, e.g.,spectroscopic, photochemical, biochemical, immunochemical, chemical, orother techniques. To illustrate, useful labels include radioisotopes,fluorescent dyes, electron-dense reagents, enzymes (as commonly used inELISAs), biotin, or haptens and proteins for which antisera ormonoclonal antibodies are available. Many of these and other labels aredescribed further herein and/or are otherwise known in the art.Exemplary primer nucleic acids of the invention, which bind to an N.gonorrhoeae nucleic acid with a sequence consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 36, a substantially identical variant thereof inwhich the variant has at least 90% sequence identity to one of SEQ IDNOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, or the variant, comprise sequences selected from SEQ ID NOS:3-27 and 37-60 and complements thereof. One of skill in the art willrecognize that, in certain embodiments, primer nucleic acids can also beused as probe nucleic acids.

A “quencher moiety” or “quencher” refers to a moiety that reduces and/oris capable of reducing the detectable emission of radiation, e.g.,fluorescent or luminescent radiation, from a source that would otherwisehave emitted this radiation. A quencher typically reduces the detectableradiation emitted by the source by at least 50%, typically by at least80%, and more typically by at least 90%. Exemplary quenchers areprovided in, e.g., U.S. Pat. No. 6,465,175, entitled “OLIGONUCLEOTIDEPROBES BEARING QUENCHABLE FLUORESCENT LABELS, AND METHODS OF USETHEREOF,” which issued Oct. 15, 2002 to horn et al., which isincorporated by reference.

The term “sample” refers to any substance containing or presumed tocontain N. gonorrhoeae and/or C. trachomatis nucleic acid including, butnot limited to, tissue or fluid isolated from one or more subjects orindividuals, in vitro cell culture constituents, as well as clinicalsamples. Exemplary samples include blood, plasma, serum, urine, synovialfluid, seminal fluid, seminal plasma, prostatic fluid, vaginal fluid,cervical fluid, uterine fluid, cervical scrapings, amniotic fluid, analscrapings, mucus, sputum, tissue, and the like.

The phrase “sample derived from a subject” refers to a sample obtainedfrom the subject, whether or not that sample undergoes one or moreprocessing steps (e.g., cell lysis, debris removal, stabilization, etc.)prior to analysis. To illustrate, samples can be derived from subjectsby scraping, venipuncture, swabbing, biopsy, or other techniques knownin the art.

The term “selectively bind” or “selective binding” in the context ofnucleic acid detection reagents refers to a nucleic acid detectionreagent that binds to an N. gonorrhoeae nucleic acid with a sequenceconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, a substantiallyidentical variant thereof in which the variant has at least 90% sequenceidentity to one of SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant to a greater extentthan the nucleic acid detection reagent binds, under the samehybridization conditions, to nucleic acids from at least three organismsselected from each of Tables X and XI.

The term “selectively detect” refers to the ability to detect an N.gonorrhoeae nucleic acid with a sequence consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 36, a substantially identical variant thereof inwhich the variant has at least 90% sequence identity to one of SEQ IDNOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, or the variant to a greater extent than nucleic acids from otherorganisms.

“Selectively hybridizing” or “selective hybridization” occurs when anucleic acid sequence hybridizes to a specified nucleic acid targetsequence to a detectably greater degree than its hybridization tonon-target nucleic acid sequences. Selectively hybridizing sequenceshave at least 50%, or 60%, or 70%, or 80%, or 90% sequence identity ormore, e.g., typically 95-100% sequence identity (i.e., complementarity)with each other.

A “sequence” of a nucleic acid refers to the order and identity ofnucleotides in the nucleic acid. A sequence is typically read in the 5′to 3′ direction.

A “sequence specific antibody” refers to an antibody that detectablybinds to nucleic acids with sequences that consist of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 36, a substantially identical variant thereof inwhich the variant has at least 90% sequence identity to one of SEQ IDNOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, or the variant.

A “sequencing reaction” refers to a reaction that includes, e.g., theuse of terminator nucleotides and which is designed to elucidate thesequence of nucleotides in a given nucleic acid.

A “set” refers to a collection of at least two things. For example, aset may include 2, 3, 4, 5, 10, 20, 50, 100, 1,000 or other number ofmolecule or sequence types. For example, certain aspects of theinvention include reaction mixtures having sets of amplicons. A “subset”refers to any portion of a set.

A “solid support” refers to a solid material that can be derivatizedwith, or otherwise attached to, a chemical moiety, such as anoligonucleotide probe or the like. Exemplary solid supports includeplates, beads, microbeads, tubes, fibers, whiskers, combs, hybridizationchips (including microarray substrates, such as those used in GeneChip®probe arrays (Affymetrix, Inc., Santa Clara, Calif., USA) and the like),membranes, single crystals, ceramic layers, self-assembling monolayers,and the like.

An oligonucleotide probe is “specific” for a target sequence if thenumber of mismatches present between the oligonucleotide and the targetsequence is less than the number of mismatches present between theoligonucleotide and non-target sequences that might be present in asample. Hybridization conditions can be chosen under which stableduplexes are formed only if the number of mismatches present is no morethan the number of mismatches present between the oligonucleotide andthe target sequence. Under such conditions, the target-specificoligonucleotide can form a stable duplex only with a target sequence.Thus, the use of target-specific primers under suitably stringentamplification conditions enables the specific amplification of thosesequences, which contain the target primer binding sites. Similarly, theuse of target-specific probes under suitably stringent hybridizationconditions enables the detection of a specific target sequence.

A “subject” refers to an organism. Typically, the organism is amammalian organism, particularly a human organism. In certainembodiments, for example, a subject is a patient suspected of having anNG and/or a CT infection.

A “subsequence” or “segment” refers to any portion of an entire nucleicacid sequence.

A “substantially identical variant” in the context of nucleic acids orpolypeptides, refers to two or more sequences that have at least 85%,typically at least 90%, more typically at least 95% nucleotide orsequence identity to one another when compared and aligned for maximumcorrespondence, as measured using, e.g., a sequence comparison algorithmor by visual inspection. The substantial identity generally exists overa region of the sequences that is at least about 15 nucleotides or aminoacids in length, more typically over a region that is at least about 20nucleotides or amino acids in length, and even more typically thesequences are substantially identical over a region of at least about 25nucleotides or amino acids in length. In some embodiments, for example,the sequences are substantially identical over the entire length of thenucleic acids or polypeptides being compared. SEQ ID NO: 36 can beconsidered as an exemplary variant of SEQ ID NO: 1.

The term “substitution” in the context of a nucleic acid sequence refersto an alteration in which at least one nucleotide of the nucleic acidsequence is replaced by a different nucleotide.

The terms “target sequence,” “target region,” and “target nucleic acid”refer to a region of a nucleic acid, which is to be amplified, detected,or otherwise analyzed.

A “terminator nucleotide” refers to a nucleotide, which uponincorporation into a nucleic acid prevents further extension of thenucleic acid, e.g., by at least one nucleotide incorporatingbiocatalyst.

A “thermostable enzyme” refers to an enzyme that is stable to heat, isheat resistant and retains sufficient catalytic activity when subjectedto elevated temperatures for selected periods of time. For example, athermostable polymerase retains sufficient activity to effect subsequentprimer extension reactions when subjected to elevated temperatures forthe time necessary to effect denaturation of double-stranded nucleicacids. Heating conditions necessary for nucleic acid denaturation arewell known in the art and are exemplified in U.S. Pat. Nos. 4,683,202and 4,683,195, which are both incorporated by reference. As used herein,a thermostable polymerase is typically suitable for use in a temperaturecycling reaction such as the polymerase chain reaction (“PCR”). For athermostable polymerase, enzymatic activity refers to the catalysis ofthe combination of the nucleotides in the proper manner to form primerextension products that are complementary to a template nucleic acid(e.g., selected subsequences of an N. gonorrhoeae or C. trachomatisgenome).

II. Overview

The invention relates to the selective detection of Neisseriagonorrhoeae. In particular, based on new detection strategies utilizingat least one of two target regions of the N. gonorrhoeae genome, N.gonorrhoeae infections can be diagnosed using the methods and reagentsdescribed herein. Each of these target regions has multiple copies inthe N. gonorrhoeae genome. Accordingly, this typically facilitates thedetection of N. gonorrhoeae in samples utilizing the approachesdescribed herein relative to techniques that target single copy regionsof the genome. In addition, the nucleic acid detection reagentsdescribed herein generally detectably bind, under selected assayconditions, to nucleotide sequences that are present in N. gonorrhoeae,but which are not present in other species, thereby minimizing theoccurrence of: e.g., false positives. This specificity is illustratedin, for example, FIGS. 5-7, 9, and 10, and the related description inthe examples provided below. Many other features of the invention arealso described herein.

To further illustrate, certain methods of the invention includecontacting or incubating nucleic acid detection reagents with nucleicacids in or from samples derived from subjects (e.g., human patientssuspected of having N. gonorrhoeae infections, etc.). In certainembodiments, target regions of the nucleic acids in the sample areamplified prior to or simultaneously with being contacted with thenucleic acid detection reagents. Nucleic acid detection reagentsdetectably bind to a nucleic acid with a sequence consisting SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36, or the variant. As described further below, SEQ ID NO:1 and SEQ ID NO: 2 are consensus sequences that correspond to tworegions of the N. gonorrhoeae genome that are targeted in the methods ofthe invention; SEQ ID NO: 36 is a variant of SEQ ID NO: 1. These methodsalso include monitoring (e.g., at a single time point, at multiplediscrete time points, continuously over a selected time period, etc.)binding between the nucleic acids and/or amplicons, and the nucleic aciddetection reagents to determine whether Neisseria gonorrhoeae is presentin the samples, e.g., to diagnose patients from which the samples werederived, to monitor courses of treatment for patients diagnosed withNeisseria gonorrhoeae infections, and/or the like.

In some embodiments, these methods further include contacting thenucleic acids and/or amplicons of the target regions with additionalnucleic acid detection reagents that detectably bind to Chlamydiatrachomatis nucleic acids. In these embodiments, the methods alsoinclude monitoring binding between the nucleic acids and/or theamplicons, and the additional nucleic acid detection reagents todetermine whether Chlamydia trachomatis is also present in the samples.Optionally, these methods are also repeated one or more times usingadditional samples (e.g., from the same subject) to monitor, e.g.,courses of treatment for subjects diagnosed with Neisseria gonorrhoeaeand/or Chlamydia trachomatis infections, the recurrence of infections,and/or the like.

Other methods of the invention include contacting or incubating nucleicacids from samples with at least a first pair of primer nucleic acidsthat include at least one nucleic acid selected from the groupconsisting of: SEQ ID NOS: 3-27 and 37-60 or complements thereof, innucleic acid amplification reactions. As described further below, SEQ IDNOS: 3-27 and 37-60 are oligonucleotides that include subsequences ofSEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 36. In addition, thesemethods also include detecting amplicons during or after theamplification reactions are performed to detect whether Neisseriagonorrhoeae is present in the samples. Optionally, these methods furtherinclude contacting the nucleic acids from the samples with at least asecond pair of primer nucleic acids that are at least partiallycomplementary to a Chlamydia trachomatis nucleic acid and detectingadditional amplicons during or after the amplification reactions areperformed to determine whether Chlamydia trachomatis is present in thesamples. These methods are also optionally repeated at selected timepoints.

In addition to compositions and reaction mixtures, the invention alsorelates to kits and systems for detecting these pathogenic agents, andto related computers and computer readable media.

III. Nucleic Acid Detection Reagents

The nucleic acid detection reagents of the invention include variousembodiments, including probe nucleic acids, primer nucleic acids, andsequence specific antibodies. Some of these nucleic acid detectionreagents target repeat 130 (also referred to herein as “NGDR9”), whichis an 806 base pair direct repeat in the N. gonorrhoeae genome that isthought to encode a protein. The N. gonorrhoeae genome includes twocopies of NGDR9, one located at nucleotide positions 458182-458988 andthe other located at nucleotide positions 1586504-1587310. A consensussequence of NGDR9 corresponds to SEQ ID NO: 1, which is shown in TableI. Although only one strand of the NGDR9 locus is shown in Table I,those of skill in the art will appreciate that SEQ ID NO: 1 identifies aregion of double stranded genomic nucleic acid, and that the sequencesof both strands are fully specified by the sequence informationprovided.

TABLE 1 SEQ ID NO: 1 CAGCCCCATC ATGATGCCGC ACGTCAGGGC TTCGTCTTCC 40GATACCTTTG CGCCAGACAA CATCCGGGCG ATGTTTTCTT 80 TTTGCGCTTT TGACCGGGCGGACAGCCGGT TCCGGTCAAC 120 GTTTCTGACC GTCCCGGCGC GTTTGACGGC GCGTTCCTGC160 CGCGTTGATT CCTTCGCCGC GCGTTTGGCG GCAAGCATCT 200 GTTTTGCCGTCGGTTTTGTT GCTACTGTTT GCATTTTGTT 240 TTCTCGATTT TTTGATGCCG TTCTCTCAATGCCCAATCAT 280 AAAGCTGTAT CTCTCACGAG GTCGCCGAAT TTAAATTGAT 320AGTTCATGTC TTGTTCCATT AATATCAAAC GCAATCTTCA 360 AACACCTCAA TTACATTTTTTAAATCGCTA ATACCATAAT 400 TTATTACATC CTTTAGAAAT TCCAAAGAGG TATCCGCTTC440 GTCTGCTTTA TCCCTAATTT CGTCTATATA ACCCTCTAAC 480 GATTCAGGCTCTTTTAATGC TTCTTTGCAT AAGTTATCTA 520 TTACCCTTAA TGCGTTTTTT ACATCTTCCAAATAGCTCAT 540 TTTTTGCTCC TTAACTCAAA ATGGGATGCT GTCGTCAACA 580TCTTCTACGG TTTATCTAAT CTGCAAATTC TTCCGCCCTT 620 CAATCTTCGC GCCTGCTACTTGCCGACCGC TTTCAATCGC 680 TTTTCTGATG GCGGTTTTGT CCGGTTCGGT TTTGACGGCC720 TCACGCATAA ATTCGGCGGG GATTTGTGCT TCGTCTAAGA 760 TCACGACGGCTTCGGATTTG CGGAACGAGG CTTTAAAAGT 800 GCCGTC 806

To illustrate, nucleic acid detection reagents comprising SEQ ID NOS:3-12, 17-20, 24-26, or complements thereof; target NGDR9 or complementsthereof. SEQ ID NOS: 3-12, 17-20, and 24-26 are shown in Table II.

TABLE II SEQ ID NO: 3 5′-CGTTCTCTCAATGCCCAATCA-3′ SEQ ID NO: 45′-AGCAGACGAAGCGGATACCTC-3′ SEQ ID NO: 5 5′-CTCTCAATGCCCAATCATAAAGC-3′SEQ ID NO: 6 5′-GTATCCGCTTCGTCTGCTTTATC-3′ SEQ ID NO: 75′-GTTTGGCGGCAAGCATCT-3′ SEQ ID NO: 8 5′-AAATGGGATGCTGTCGTCAA-3′ SEQ IDNO: 9 5′-GGCAAGCTTGTTTGGCGGCAAGCATCT-3′ SEQ ID NO: 105′-GGCGGATCCTTGACGACAGCATCCCATTT-3′ SEQ ID NO: 115′-AAACGCAATCTTCAAACACCTCA-3′ SEQ ID NO: 12 5′-TTTGACGGCCTCACGCATAA-3′SEQ ID NO: 17 5′-CGAGGTCGCCGAATTTAAATTGATAGTT-3′ SEQ ID NO: 185′-AACTATCAATTTAAATTCGGCGACCTCG-3′ SEQ ID NO: 195′-CGAGGTCGCCGAATTTAAATTGATAGTTCA-3′ SEQ ID NO: 205′-TGAACTATCAATTTAAATTCGGCGACCTCG-3′ SEQ ID NO: 245′-GATAAAGCAGACGAAGCGGATAC-3′ SEQ ID NO: 25 5′-TTGACGACAGCATCCCATTT-3′SEQ ID NO: 26 5′-TTATGCGTGAGGCCGTCAAA-3′

Other nucleic acid detection reagents of the invention target repeat 116(also referred to herein as “NGDR33”), which is an 1142 base pair directrepeat in the N. gonorrhoeae genome that is thought to encode apolypeptides. The N. gonorrhoeae genome includes two copies of NGDR33,one located at nucleotide positions 491768-492910 and the other locatedat nucleotide positions 1606987-1608129. A consensus sequence of NGDR33corresponds to SEQ ID NO: 2, which is shown in Table III. Although onlyone strand of the NGDR33 locus is shown in Table III, those of skill inthe art will appreciate that SEQ ID NO: 2 identifies a region of doublestranded genomic nucleic acid, and that the sequences of both strandsare fully specified by the sequence information specified.

TABLE III SEQ ID NO: 2 ACGCCGTGGT GCGGCCTGTT TGTCGGATAC TGCCTGGGCA 40AAAGCGGACG CGCGGTCATC AGGGACTGGT ATCGCGCCAA 80 AGCCTGGTCA ATGTCGGGTTTGACGAAACT CGAAGCCCCC 120 GCATACGGCT GCATCGCGGT CAAACCGCGC CGGGGCGGCG160 GACACGTGTT CTTCGTTGTC GGCAAAGACG CGGAAGGCAG 200 AATCTTGGGCTTGGGCGGCA ATCAGGGCAA TATGGTATCC 240 ATCATCCCGT TTGACCCTGC GGACATTGACGGCTACTTCT 280 GGCCGTCCAA GCTGATTGGC GGCAAAGCCG TGCCTTCGTC 320CCCCGCCGAA GGGCGTTACC GGTTGTCGGA CGTTGCCGCC 360 ACGGCGAAAC AGGGCGCGGGCGAGGCGTAA ATGATTGGGG 400 CTTTGCTGAA AAATTGGAAG CCGCTGCTTA TTTTGTCCGC440 AATCGCGTTC TTCGCCGTTT CTTGGCAGCT GGACAGGGCG 480 GCGCAATACCGTCGCGGATA CGGTGCGGCG GTGTCGGAGG 520 TTTCGGAACG CCTCAAAGCC GCCGCGGTCGAACACGCCGA 560 ACACGCCCGC AAATCGTCCG CCGCGTATCA GGCGCAAAAG 600GCGGCGCGCG AGGAAAAAGA AAGGGTGCGC TATGTGGAAA 640 CGCTTAAAAT CATTGAAAAACCTGTGTACC GCAATGCCTG 680 TTTTGATGCT GACGGCGTGC GCGAACTCAA CGCCGCCGTT720 GACGACGGCG GTTAAGCCGC CCGCCGATTT GGTGCGGCCC 760 TGCCCGAAACTGCCGCACCT TGAAGGGAAC ACGGGCGCGG 800 ACGTGCTGCC GTGGGCCCTG AAGGCGGCCGGTATGTATAA 840 CGACTGCAGG GCGCGGCACG GCGCGCTGGT ACGGGCGTTG 880GGCGCGGATT GAGTTGTCAA CCGGAAGTTT GCAACCGAAC 920 CGTCGGTTCC GGGTTGGCGGCCGCATCGGG GGAAGTGTCG 960 GCATTCCCCC CGATTTTTTA CATATCGGGC GGACGCGGCA1000 AATTTTTGCC GTTTTGTTTG CGCGAAGGGG GCGTTATACA 1040 AAATTATCAGGCGCACCAAT AATGGGCGGA AATGAAAATG 1080 CCGTACCGAT CCGGACAACA ACCGATGCCGCACCCTGCGG 1120 GCAGGCTTCG CACTCTGAAA GG 1142

For example, nucleic acid detection reagents corresponding to SEQ IDNOS: 13-16, 21-23, 27, or complements thereof target NGDR33 orcomplements thereof SEQ ID NOS: 13-16, 21-23, and 27 are shown in TableIV.

TABLE IV SEQ ID NO: 13 5′-TCAATGTCGGGTTTGACGAA-3′ SEQ ID NO: 145′-AACGTCCGACAACCGGTAAC-3′ SEQ ID NO: 15 5′-AATGTCGGGTTTGACGAAACTC-3′SEQ ID NO: 16 5′-GTTACCGGTTGTCGGACGTT-3′ SEQ ID NO: 215′-GCGGCAATCAGGGCAATATGGTAT-3′ SEQ ID NO: 225′-ATACCATATTGCCCTGATTGCCGC-3′ SEQ ID NO: 235′-GGCGGCAATCAGGGCAATATGGTAT-3′ SEQ ID NO: 27 5′-AACGTCCGACAACCGGTAAC-3′

In certain embodiments where NGDR9 is targeted, probes and/or primersoptionally detectably bind to a nucleic acid segment that comprises oneor more nucleotide positions of SEQ ID NO: 1 selected from the groupconsisting of: 259, 260, 262, 264, 265, 266, 268, 269, 273, 275, 276,277, 279, 297, 298, 300, 301, 302, 303, 304, 305, 306, 308, 313, 314,315, 316, 317, 318, 320, 321, 325, 326, 428, 429, 431, 432, 433, 434,435, 440, 441, and 447. These nucleotide positions, which arehighlighted and underlined in Table V, denote certain exemplarymismatches with the sequence of Brucella suis 1330 chromosome I section155 (GenBank® accession number AE014469) that were identified in analignment of the sequences of NGDR9 and B. suis 1330 chromosome Isection 155. Other mismatches with this sequence from the Brucella suisgenome are illustrated in FIG. 4. This sequence of the B. suis genomehas a higher level of identity with NGDR9 than sequences from otherbacterial species. An alignment of the sequence of NGDR9 with this B.suis sequence is described further in an example provided below.

TABLE IV SEQ ID NO: 1 CAGCCGCATC ATGATGCCGC ACGTCAGGGC TTCGTCTTCC 40GATACCTTTG CGCCAGACAA CATCCGGGCG ATGTTTTCTT 80 TTTGCGCTTT TGACCGGGCGGACAGCCGGT TCCGGTCAAC 120 GTTTCTGACC GTCCCGGCGC GTTTGACGGC GCGTTCCTGC160 CGCGTTGATT CCTTCGCCGC GCGTTTGGCG GCAAGCATCT 200 GTTTTGCCGTCGGTTTTGTT GCTACTGTTT GCATTTTGTT 240 TTCTCGATTT TTTCATGC CG  T T C TCT CAA T GC C C AAT C A T 280 AAAGCTGTAT CTCTCA CG A G   GTCGCC G A AT TTAAATTG A T 320 A GTT CA TGTC TTGTTCCATT AATATCAAAC GCAATCTTCA 360AACACCTCAA TTACATTTTT TAAATCGCTA ATACCATAAT 400 TTATTACATC CTTTAGAAATTCCAAAG AG G TATCC GCTT C 440 G TCTGC T TTA TCCCTAATTT CGTCTATATAACCCTCTAAC 480 GATTCAGGCT CTTTTAATGC TTCTTTGCAT AAGTTATCTA 520TTACCCTTAA TGCGTTTTTT ACATCTTCCA AATAGCTCAT 540 TTTTTGCTCC TTAACTCAAAATGGGATGCT GTCGTCAACA 580 TCTTCTACGG TTTATCTAAT CTGCAAATTC TTCCGCCCTT620 CAATCTTCGC GCCTGCTACT TGCCGACCGC TTTCAATCGC 680 TTTTCTGATGGCGGTTTTGT CCGGTTCGGT TTTGACGGCC 720 TCACGCATAA ATTCGGCGGG GATTTGTGCTTCGTCTAAGA 760 TCACGACGGC TTCGGATTTG CGGAACGAGG CTTTAAAAGT 800 GCCGTC806

In some embodiments were NGDR33 is targeted, probes and/or primersoptionally detectably bind to a nucleic acid segment that comprises oneor more nucleotide positions of SEQ ID NO: 2 selected from the groupconsisting of: 89, 90, 91, 92, 95, 98, 101, 105, 106, 107, 216, 217,220, 222, 223, 225, 233, 235, 236, 238, 335, 336, 337, 338, 339, 342,345, 346, and 351. These nucleotide positions, which are highlighted andunderlined in Table VI, denote some exemplary mismatches with thesequence of Neisseria meningitidis serogroup B strain MC58 section 77(GenBank® accession number AE002435) that were identified in analignment of the sequences of NGDR33 and N. meningitidis serogroup Bstrain MC5S8 section 77. Other mismatches with this sequence from the N.meningitidis genome are illustrated in FIG. 8. This sequence of the N.meningitidis genome has a higher level of identity with NGDR33 thansequences from other bacterial species. An alignment of the sequence ofNGDR33 with this N. meningitidis sequence is described further in anexample provided below.

TABLE VI SEQ ID NO: 2 ACGCCGTGGT GCGGCCTGTT TGTCGGATAC TGCCTGGGCA 40AAAGCGGACG CGCGGTCATC AGGGACTGGT ATCGCGCCAA 80 AGCCTGGT CA   AT GT C GGG TT T GAC GAA ACT CGAAGCCCCC 120 GCATACGGCT GCATCGCGGT CAAACCGCGCCGGGGCGGCG 160 GACACGTGTT CTTCGTTGTC GGCAAAGACG CGGAAAGCAG 200AATCTTGGGC TTGGG CG GC A  A TC A G GGCAA TA T G GT A T CC 240 ATCATCCCGTTTGACCCTGC GGACATTGAC GGCTACTTCT 280 GGCCGTCCAA GCTCATTGGC GGCAAAGCCGTGCCTTCGTC 320 CCCCGCCGAA GGGC GTTAC C G G TT GT CGGA C GTTGCCGCC 360ACGGCGAAAC AGGGCGCGGG CGAGGCGTAA ATGATTGGGG 400 CTTTGCTGAA AAATTGGAAGCCGCTGCTTA TTTTGTCCGC 440 AATCGCGTTC TTCGCCGTTT CTTGGCAGCT GGACAGGGCG480 GCGCAATACC GTCGCGGATA CGGTGCGGCG GTGTCGGAGG 520 TTTCGGAACGCCTCAAAGCC GCCGCGGTCG AACACGCCGA 560 ACACGCCCGC AAATCGTCCG CCGCGTATCAGGCGCAAAAG 600 GCGGCGCGCG AGGAAAAAGA AAGGGTGCGC TATGTGCAAA 640CGCTTAAAAT CATTGAAAAA CCTGTGTACC GCAATGCCTG 680 TTTTGATGCT GACGGCGTGCGCGAACTCAA CGCCGCCGTT 720 GACGACGGCG GTTAAGCCGC CCGCCGATTT GGTGCGGCCC760 TGCCCGAAAC TGCCGCACCT TGAAGGGAAC ACGGGCGCGG 800 ACGTGCTGCCGTGGGCCCTG AAGGCGGCCG GTATGTATAA 840 CGACTGCAGG GCGCGGCACG GCGCGCTGGTACGGGCGTTG 880 GGCGCGGATT GAGTTGTCAA CCGGAAGTTT GCAACCGAAC 920CGTCGGTTCC GGGTTGGCGG CCGCATCGGG GGAAGTGTCG 960 GCATTCCCCC CGATTTTTTACATATCGGGC GGACGCGGCA 1000 AATTTTTGCC GTTTTGTTTG CGCGAAGGGG GCGTTATACA1040 AAATTATCAG CCGCACCAAT AATGGGCGGA AATGAAAATG 1080 CCGTACCGATCCGGACAACA ACCGATGCCG CACCCTGCGG 1120 GCAGGCTTCG CACTCTGAAA GG 1142

Other nucleic acid detection reagents of the invention target avariation sequence of repeat 130 (also referred to herein as“NGDR9Var”). A consensus sequence of NGDR9Var corresponds to SEQ TD NO:36, which is shown in Table A. Although only one strand of the NGDR9Varlocus is shown in Table A, those of skill in the art will appreciatethat SEQ ID NO: 36 identifies a region of double stranded genomicnucleic acid, and that the sequences of both strands are fully specifiedby the sequence information specified.

TABLE A SEQ ID NO: 36 CAGCCGCATC ATGATGCCGC ACGTCAGGGC TTCGTCTTCC 40GATACCTTTG CGCCCGACAA CATCCGGGCG ATGTTTTCTT 80 TTTGCGCTTT TGACCGGGCGGACAGCCGGT TCCGGTCAAC 120 GTTTCTGACC GTCCCGGCGC GTTTGACGGC GCGTTCCTGC160 CGCGTTGATT CCTTCGCCGC GCGTTTGGCG GCAAGCATCT 200 GTTTTGCCGTCGGTTTTGTT GTTGCCGTTT GCATTTTTAC 240 CTCCTTTTAA AACTGTTTCG ACAGGCTTGCGAACGGCCTT 280 CCCGTTCACT TCCCGCGCCT GCCTGATTCC TACGGTGCGG 320ATGTCGGGAT TGCCCGGCGG CAGCCTGACA AACCCTTCGG 360 CGGCCCCTAT GGTTGGATATCCGGGGCTGA TTCGGCAGCG 400 GCTGTTGCTT GGCCGCTCCC GTCCGCGCCG GTCTGCGTAC440 CGCTTGGAAT CCTCTTTGTC GCAAACAGGC CGCCGCCGTT 480 TTTCTTCTTCGGGACGATGT TTTCCAAAGG CTGCGAACAT 520 GGCGGCCCCT GTTTATCTAA TCTGCAAATTCTTCCGCTCT 540 TCAATCTTCG CGCCTGCTAC TTGCCGACCG CTTTCAATCG 580CTTTTCTGAT GGCGGTTTTG TCCGGTTCGG TTTTGACGGC 620 CTCACGCATA AATTCGGCGGGGATTTGTGC TTCGTCTAAG 680 ATCACGACGG CTTCGGATTT GCGGAACGAG GCTTTAAAAG720 TGCCGTC 760

To illustrate, nucleic acid detection reagents comprising SEQ ID NOS:37-45, or complements thereof, target NGDR9Var or complements thereof.SEQ ID NOS: 37-45 are shown in Table B:

TABLE B SEQ ID NO: 37 5′-GTTTCGACAGGCTTGCGAAC-3′ SEQ ID NO: 385′-GTTTCGACAGGCTTGCGAA-3′ SEQ ID NO: 39 5′-TTTGTTGTTGCCGTTTGCA-3′ SEQ IDNO: 40 5′-CCTGTTTGCGACAAAGAGGA-3′ SEQ ID NO: 415′-TGCGACAAAGAGGATTCCAA-3′ SEQ ID NO: 42 5′-TTCCCGCGCCTGCCTGATTCCTA-3′SEQ ID NO: 43 5′-ACATCCGCACCGTAGGAATCAGGCA-3′ SEQ ID NO: 445′-AATCCCGACATCCGCACCGTAGGAATCA-3′ SEQ ID NO: 455′-AATCCCGACATCCGCACCGTAGGAAT-3′

Other nucleic acid detection reagents of the invention target both NGDR9and NGDR9Var. To illustrate, nucleic acid detection reagents comprisingSEQ ID NOS: 46-60, or complements thereof, target NGDR9 and NGDR9Var orcomplements thereof. These sequences can also be termed “universal”sequences. SEQ ID NOS: 46-60 are shown in Table C:

TABLE C SEQ ID NO: 46 5′-CGTTTGGCGGCAAGCATC-3′ SEQ ID NO: 475′-GCGGCAAGCATCTGTTTTGC-3′ SEQ ID NO: 48 5′-GTAGCAGGCGCGAAGATTGAA-3′ SEQID NO: 49 5′-AGTAGCAGGCGCGAAGATTGA-3′ SEQ ID NO: 505′-GGGCGGAAGAATTTGCAGATTA-3′ SEQ ID NO: 51 5′-GCCATCAGAAAAGCGATTGAAA-3′SEQ ID NO: 52 5′-GGACAAAACCGCCATCAGAAA-3′ SEQ ID NO: 535′-TAATCTGCAAATTCTTCCGCCCTTCAATCTT-3′ SEQ ID NO: 545′-TTTATCTAATCTGCAAATTCTTCCGCCCTTC A-3′ SEQ ID NO: 555′-CAAATTCTTCCGCCCTTCAATCTTCGC-3′ SEQ ID NO: 565′-CTTCAATCTTCGCGCCTGCTACTTGCC-3′ SEQ ID NO: 575′-AAGATTGAAGGGCGGAAGAATTTGCAGATTA-3′ SEQ ID NO: 585′-GCGAAGATTGAAGGGCGGAAGAATTTG-3′ SEQ ID NO: 595′-TCAGGGCTTCGTCTTCCGA-3′ SEQ ID NO: 60 5′-TTTAAAGCCTCGTTCCGCAA-3′

As mentioned above, nucleic acid detection reagents compriseoligonucleotides (e.g., probe nucleic acids, primer nucleic acids, etc.)in certain embodiments of the invention. Although other lengths areoptionally utilized, oligonucleotides generally comprise sequences thatare typically between about 8 and about 100 nucleotides in length, moretypically between about 10 and about 75 nucleotides in length, stillmore typically between about 12 and about 50 nucleotides in length, andeven more typically between about 15 and about 35 nucleotides in length(e.g., about 20, about 25, or about 30 nucleotides in length). Methodsof preparing oligonucleotides, such as nucleic acid synthesis, aredescribed further below.

Various approaches can be utilized by one of skill in the art to designoligonucleotides (e.g., substantially identical variants of nucleicacids having sequences selected from SEQ ID NOS: 3-27 and 37-60 orcomplements thereof) that selectively bind to NGDR9 and/or NGDG9Var, orNGDR33, which oligonucleotides can be used to detect N. gonorrhoeae. Toillustrate, the DNAstar software package available from DNASTAR, Inc.(Madison, Wis.) can be used for sequence alignments. For example,nucleic acid sequences for NGDR9 and B. suis or NGDR33 and N.meningitidis can be uploaded into DNAstar EditSeq program as individualfiles. Pairs of sequence files (e.g., NGDR9 and B. suis) can be openedin the DNAstar MegAlign sequence alignment program and the Clustal Wmethod of alignment can be applied. The parameters used for Clustal Walignments are optionally the default settings in the software. MegAligntypically does not provide a summary of the percent identity between twosequences. This is generally calculated manually. From the alignments,regions having, e.g., less than 85% identity with one another aretypically identified and oligonucleotide sequences in these regions canbe selected. Many other sequence alignment algorithms and softwarepackages are also optionally utilized. Sequence alignment algorithms arealso described in, e.g., Mount, Bioinformatics: Sequence and GenomeAnalysis, Cold Spring Harbor Laboratory Press (2001), and Durbin et al.,Biological Sequence Analysis Probabilistic Models of Proteins andNucleic Acids, Cambridge University Press (1998), which are bothincorporated by reference.

To further illustrate, optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman(1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA85:2444, which are each incorporated by reference, and by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group(Madison, Wis.), or by even by visual inspection.

Another example algorithm that is suitable for determining percentsequence identity is the BLAST algorithm, which is described in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410, which is incorporatedby reference. Software for performing versions of BLAST analyses ispublicly available through the National Center for BiotechnologyInformation on the world wide web at ncbi.nlm.nih.gov/ as of Mar. 12,2004. This algorithm involves first identifying high scoring sequencepairs (HSPs) by identifying short words of length W in the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as the neighborhood word score threshold(Altschul et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915, which is incorporated by reference).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5787, which is incorporated by reference). One measureof similarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a nucleic acid is considered similar to areference sequence (and, therefore, homologous) if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, or less than about 0.01, and oreven less than about 0.001.

An additional example of a useful sequence alignment algorithm isPILEUP. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle (1987) J. Mol. Evol. 35:351-360, which isincorporated by reference. The method used is similar to the methoddescribed by Higgins & Sharp (1989) CABIOS 5:151-153, which isincorporated by reference. The program can align, e.g., up to 300sequences of a maximum length of 5,000 letters. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This clustercan then be aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences can be aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program can also be used to plot a dendogram or treerepresentation of clustering relationships. The program is run bydesignating specific sequences and their amino acid or nucleotidecoordinates for regions of sequence comparison.

The probes and primers of the invention optionally include one or morelabels, which are described further below. In addition, probes andprimers optionally include various other modifications, such as modifiednucleotides that alter hybridization melting temperatures, restrictionsite linkers to facilitate amplicon cloning, modifier groups thatincrease the specificity of nucleic acid amplification reactions, and/orthe like. For example, certain modified nucleotides that increasenucleic acid hybridization melting temperatures are optionally includedto permit the use of smaller probes and primers, such as those includingbetween about 8 and about 14 nucleotides. Examples of these modifiedoligonucleotides include those having one or more LNA™ monomers.Nucleotide analogs such as these are described further in, e.g., U.S.Pat. No. 6,639,059, entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,”issued Oct. 28, 2003 to Kochkine et al., U.S. Pat. No. 6,303,315,entitled “ONE STEP SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS INCOMPLEX BIOLOGICAL SAMPLES,” issued Oct. 16, 2001 to Skouv, and U.S.Pat. Application Pub. No. 2003/0092905, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al. that published May 15,2003, which are each incorporated by reference. Oligonucleotidescomprising LNA™ monomers are commercially available through, e.g.,Exiqon A/S (Vedbæk, DK). Additional probe and primer modifications arereferred to herein, including in the definitions provided above.

In certain embodiments, the nucleic acid detection reagents utilized asdescribed herein are sequence specific antibodies that target SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 36, a substantially identical variantthereof in which the variant has at least 90% sequence identity to oneof SEQ ID NOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 36, or the variant. SEQ ID NO: 1 and SEQ ID NO: 2 and SEQID NO: 36 are described further above and are provided in Tables I andII and A, respectively.

Antibodies suitable for use in these embodiments of invention may beprepared by conventional methodology and/or by genetic engineering.Antibody fragments may be genetically engineered, e.g., from thevariable regions of the light and/or heavy chains (V_(H) and V_(L)),including the hypervariable regions, or from both the V_(H) and V_(L)regions. For example, the term “antibodies” as used herein includespolyclonal and monoclonal antibodies and biologically active fragmentsthereof including among other possibilities “univalent” antibodies(Glennie et al. (1982) Nature 295:712); Fab proteins including Fab′ andF(ab′)₂ fragments whether covalently or non-covalently aggregated; lightor heavy chains alone, typically variable heavy and light chain regions(V_(H) and V_(L) regions), and more typically including thehypervariable regions (otherwise known as the complementaritydetermining regions (CDRs) of the V_(H) and V_(L) regions); F_(c)proteins; “hybrid” antibodies capable of binding more than one antigen;constant-variable region chimeras; “composite” immunoglobulins withheavy and light chains of different origins; “altered” antibodies withimproved specificity and other characteristics as prepared by standardrecombinant techniques, by mutagenic techniques, or other directedevolutionary techniques known in the art.

The sequence specific antibodies utilized as described herein may belabeled or unlabeled. Suitable labels include, e.g., radionuclides,enzymes, coenzymes, fluorescent dyes, chemiluminescent dyes, chromogens,enzyme substrates or co-factors, enzyme inhibitors, free radicals, andthe like. Such labeled reagents may be used in a variety of well knownassays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,fluorescent immunoassays, and the like. See, e.g., U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; and 4,233,402, which are eachincorporated by reference. Additional labels are described furtherherein.

In some embodiments, transcribed RNAs and/or translated proteins encodedby NGDR9 and NGDR33 and NGDR9Var are targeted for detection. Manytechniques for detecting RNAs and/or proteins are known in the art. Forexample, probe and primer nucleic acids of the invention can be adaptedfor use in reverse transcription-polymerase chain reaction (RT-PCR)assays for the detection of NGDR9 and/or NGDR9Var, or NGDR33transcription products. Moreover, various electrophoretic assays (e.g.,SDS-PAGE or the like), immunoassays, mass spectrometric assays (e.g.,matrix assisted laser desorption/ionization (MALDI)-based analyses,surface enhanced laser desorption/ionization (SELDI)-based assays,etc.), and/or other approaches can be used to detect proteins encoded byNGDR9 and/or NGDR9Var, or NGDR33. Many of these and other suitable RNAand protein detection methods are described in the references citedherein.

In practicing the present invention, many conventional techniques inmolecular biology and recombinant DNA are optionally used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger), DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D.N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal,1984, A Practical Guide to Molecular Cloning; the series, Methods inEnzymology (Academic Press, Inc.); Gene Transfer Vectors for MammalianCells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory); Methods in Enzymology Vol. 154 and Vol. 155 (Wu andGrossman, and Wu, eds., respectively), all of which are incorporated byreference.

IV. Sequence Variations

Numerous nucleic acid and polypeptide sequences are within the scope ofthe present invention. To illustrate, FIG. 1 shows a sequence alignmentof the Neisseria gonorrhoeae Direct Repeat 9 (NGDR9) sequence with thesequences of at least portions of amplicons of genomic DNA from variousN. gonorrhoeae strains. More specifically, genomic DNA from 5 N.gonorrhoea strains (i.e., NG strains 1117, 1120, 6346, 6359, and 6364)was amplified and sequenced with primer nucleic acids corresponding toDK101 (SEQ ID NO: 7) and DK102R (SEQ ID NO: 25). The location of theoligonucleotides DK101 and the complement to DK102R (i.e., DK102 (SEQ IDNO: 8)) and the oligonucleotides NG519 (SEQ ID NO: 5) and NG514 (SEQ IDNO: 6) are underlined in the sequence of NGDR9 shown in FIG. 1. Inaddition, the majority or consensus sequence between the DR9 sequenceand the five N. gonorrhoeae strains is also indicated.

To further illustrate, certain exemplary NGDR9-related nucleic acidsequence variations are associated with Gene ID numbers NG0465, NG0466,NG0467, IGR0389, IGR0390, NG1616, NG1617, NG1618, IGR1318, and IGR1319,and certain exemplary NGDR33-related nucleic acid sequence variationsare associated with Gene ID numbers NG0518, NG0519, NG0520, IGR0430,IGR0431, NG1649, NG1650, IGR1345, and IGR1346, all of which are providedon the world wide web at stdgen.lanl.gov as of Mar. 12, 2004. However,the NGDR9Var sequence (SEQ ID NO: 36) has not been found in any publiclyaccessible databases as of Aug. 1, 2007. SEQ ID NO: 36 was surprisinglyidentified during an extensive sequence analysis study of theNGDR9Region.

Silent Variations

It will be appreciated by those skilled in the art that due to thedegeneracy of the genetic code, a multitude of nucleic acids sequencesencoding NGDR9 and NGDR33 and NGDR9Var polypeptides may be produced,some of which may bear minimal sequence homology to the nucleic acidsequences explicitly disclosed herein. Exemplary NGDR9 polypeptides areassociated with Gene ID numbers NG0465, NG0466, NG0467, NG1616, NG1617,and NG1618, and exemplary NGDR33 polypeptides are associated with GeneID numbers NG0518, NG0519, NG0520, NG1649, and NG1650, all of which areprovided on the world wide web at stdgen.lanl.gov as of Mar. 12, 2004.

TABLE VII Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For instance, inspection of the codon table (Table VII) shows thatcodons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acidarginine. Thus, at every position in the nucleic acids of the inventionwhere an arginine is specified by a codon, the codon can be altered toany of the corresponding codons described above without altering theencoded polypeptide. It is understood that U in an RNA sequencecorresponds to T in a DNA sequence.

Such “silent variations” are one species of “conservatively modifiedvariations”, discussed below. One of skill will recognize that eachcodon in a nucleic acid (except AUG, which is ordinarily the only codonfor methionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention provides each and every possiblevariation of nucleic acid sequence encoding NGDR9 and NGDR33 andNGDR9Var polypeptides that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code (e.g., as set forth in Table 1)as applied to the nucleic acid sequences encoding NGDR9 and NGDR33 andNGDR9Var polypeptides. All such variations of every nucleic acid hereinare specifically provided and described by consideration of the sequencein combination with the genetic code.

Conservative Variations

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence refers to thosenucleic acids, which encode identical or essentially identical aminoacid sequences, or, where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. One of skill willrecognize that individual substitutions, deletions or additions whichalter, add or delete a single amino acid or a small percentage of aminoacids (typically less than 5%, more typically less than 4%, 2% or 1%) inan encoded sequence are “conservatively modified variations” where thealterations result in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table VIII sets forth six groups, whichcontain amino acids that are “conservative substitutions” for oneanother.

TABLE VIII Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Thus, “conservatively substituted variations” of a NGDR9 or a NGDR33 ora NGDR9Var polypeptide referred to herein include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup.

The addition of sequences that do not alter the encoded activity of anucleic acid molecule, such as the addition of a non-functionalsequence, is a conservative variation of the basic nucleic acid.

One of skill will appreciate that many conservative variations of thenucleic acids described herein yield a functionally identical nucleicacid. For example, as discussed above, owing to the degeneracy of thegenetic code, “silent substitutions” (i.e., substitutions in a nucleicacid sequence which do not result in an alteration in an encodedpolypeptide) are an implied feature of every nucleic acid sequence,which encodes an amino acid. Similarly, “conservative amino acidsubstitutions,” in one or a few amino acids in an amino acid sequenceare substituted with different amino acids with highly similarproperties, are also readily identified as being highly similar to adisclosed construct. Such conservative variations of each disclosedsequence are a feature of the present invention.

V. Probe and Primer Synthesis

The oligonucleotide probes and primers of the invention are optionallyprepared using essentially any technique known in the art. In certainembodiments, for example, the oligonucleotide probes and primersdescribed herein are synthesized chemically using essentially anynucleic acid synthesis method, including, e.g., according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts. 22(20):1859-1862, which isincorporated by reference, or another synthesis technique known in theart, e.g., using an automated synthesizer, as described inNeedham-VarDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168, whichis incorporated by reference. A wide variety of equipment iscommercially available for automated oligonucleotide synthesis.Multi-nucleotide synthesis approaches (e.g., tri-nucleotide synthesis,etc.) are also optionally utilized. Moreover, the primer nucleic acidsdescribed herein optionally include various modifications. In certainembodiments, for example, primers include restriction site linkers,e.g., to facilitate subsequent amplicon cloning or the like. To furtherillustrate, primers are also optionally modified to improve thespecificity of amplification reactions as described in, e.g., U.S. Pat.No. 6,001,611, entitled “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS,”issued Dec. 14, 1999 to Will, which is incorporated by reference.Primers and probes can also be synthesized with various othermodifications as described herein or as otherwise known in the art.

Essentially any label is optionally utilized to label the nucleic aciddetection reagents of the invention. In some embodiments, for example,the label comprises a fluorescent dye (e.g., a rhodamine dye (e.g., R6G,R110, TAMRA, ROX, etc.), a fluorescein dye (e.g., JOE, VIC, TET, HEX,FAM, etc.), a halofluorescein dye, a cyanine dye (e.g., CY3, CY3.5, CY5,CY5.5, etc.), a BODIPY® dye (e.g., FL, 530/550, TR, TMR, etc.), an ALEXAFLUOR® dye (e.g., 488, 532, 546, 568, 594, 555, 653, 647, 660, 680,etc.), a dichlororhodamine dye, an energy transfer dye (e.g., BIGDYE™ v1 dyes, BIGDYE™ v 2 dyes, BIGDYE™ v 3 dyes, etc.), Lucifer dyes (e.g.,Lucifer yellow, etc.), CASCADE BLUE®, Oregon Green, and the like.Additional examples of fluorescent dyes are provided in, e.g., Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Products,Ninth Ed. (2003) and the updates thereto, which are each incorporated byreference. Fluorescent dyes are generally readily available from variouscommercial suppliers including, e.g., Molecular Probes, Inc. (Eugene,Oreg.), Amersham Biosciences Corp. (Piscataway, N.J.), AppliedBiosystems (Foster City, Calif.), etc. Other labels include, e.g.,biotin, weakly fluorescent labels (Yin et al. (2003) Appl EnvironMicrobiol. 69(7):3938, Babendure et al. (2003) Anal. Biochem. 317(1):1,and Jankowiak et al. (2003) Chem Res Toxicol. 16(3):304),non-fluorescent labels, colorimetric labels, chemiluminescent labels(Wilson et al. (2003) Analyst. 128(5):480 and Roda et al. (2003)Luminescence 18(2):72), Raman labels, electrochemical labels,bioluminescent labels (Kitayama et al. (2003) Photochem Photobiol.77(3):333, Arakawa et al. (2003) Anal. Biochem. 314(2):206, and Maeda(2003) J. Pharm. Biomed. Anal. 30(6): 1725), and an alpha-methyl-PEGlabeling reagent as described in, e.g., U.S. Provisional PatentApplication No. 60/428,484, filed on Nov. 22, 2002, which references areeach incorporated by reference. Nucleic acid labeling is also describedfurther below.

In addition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asThe Midland Certified Reagent Company, The Great American Gene Company,ExpressGen Inc., Operon Technologies Inc., Proligo LLC, and many others.

VI. Sample Preparation and Nucleic Acid Amplification

Samples are generally derived or isolated from subjects, typicallymammalian subjects, more typically human subjects, suspected of havingan N. gonorrhoeae and/or C. trachomatis infections. Exemplary samples orspecimens include blood, plasma, serum, urine, synovial fluid, seminalfluid, seminal plasma, prostatic fluid, vaginal fluid, cervical fluid,uterine fluid, cervical scrapings, amniotic fluid, anal scrapings,mucus, sputum, tissue, and the like. Essentially any technique foracquiring these samples is optionally utilized including, e.g.,scraping, venipuncture, swabbing, biopsy, or other techniques known inthe art. To further illustrate, throat swabs are taken from subjects incertain embodiments, e.g., as part of screens for gonococcal pharyngitisor the like.

Methods of storing specimens, culturing cells, isolating and preparingnucleic acids from these sources are generally known in the art and manyof these are described further in the references and/or examplesprovided herein.

To further illustrate, prior to analyzing the target nucleic acidsdescribed herein, those nucleic acids may be purified or isolated fromsamples that typically include complex mixtures of different components.Cells in collected samples are typically lysed to release the cellcontents. For example, N. gonorrhoeae and other cells in the particularsample can be lysed by contacting them with various enzymes, chemicals,and/or lysed by other approaches known in the art, which degrade, e.g.,bacterial cell walls. In some embodiments, nucleic acids are analyzeddirectly in the cell lysate. In other embodiments, nucleic acids arefurther purified or extracted from cell lysates prior to detection.Essentially any nucleic acid extraction methods can be used to purifynucleic acids in the samples utilized in the methods of the presentinvention. Exemplary techniques that can be used to purifying nucleicacids include, e.g., affinity chromatography, hybridization to probesimmobilized on solid supports, liquid-liquid extraction (e.g.,phenol-chloroform extraction, etc.), precipitation (e.g., using ethanol,etc.), extraction with filter paper, extraction with micelle-formingreagents (e.g., cetyl-trimethyl-ammonium-bromide, etc.), binding toimmobilized intercalating dyes (e.g., ethidium bromide, acridine, etc.),adsorption to silica gel or diatomic earths, adsorption to magneticglass particles or organo silane particles under chaotropic conditions,and/or the like. Sample processing is also described in, e.g., U.S. Pat.Nos. 5,155,018, 6,383,393, and 5,234,809, which are each incorporated byreference.

To further exemplify, unmodified nucleic acids can bind to a materialwith a silica surface. Many of these processes that are optionallyadapted for use in the performing the methods of the present inventionare described in the art. To illustrate, Vogelstein et al. (1979) Proc.Natl. Acad. Sci. USA 76:615-619, which is incorporated by reference,describes the purification of nucleic acids from agarose gels in thepresence of sodium iodide using ground flint glass. Marko et al. (1982)Anal. Biochem. 121:382-387, which is incorporated by reference,describes the purification of nucleic acids from bacteria on glass dustin the presence of sodium perchlorate. In DE-A 3734442, which isincorporated by reference, nucleic acids are isolated on glass fiberfilters. The nucleic acids bound to these glass fiber filters are washedand then eluted with a methanol-containing Tris/EDTA buffer. A similarprocedure is described in Jakobi et al. (1988) Anal. Biochem.175:196-201, which is incorporated by reference. In particular, Jakobiet al. describes the selective binding of nucleic acids to glasssurfaces in chaotropic salt solutions and separating the nucleic acidsfrom contaminants, such as agarose, proteins, and cell residue. Toseparate the glass particles from the contaminants, the particles can becentrifuged or fluids can be drawn through the glass fiber filters. Inaddition, the use of magnetic particles to immobilize nucleic acidsafter precipitation by adding salt and ethanol is described in, e.g.,Alderton et al. (1992) Anal. Biochem. 201:166-169 and PCT/GB91/00212,which are both incorporated by reference. In this procedure, the nucleicacids are agglutinated along with the magnetic particles. Theagglutinate is separated from the original solvent by applying amagnetic field and performing one or more washing steps. After at leastone wash step, the nucleic acids are typically dissolved in a Trisbuffer.

Magnetic particles in a porous glass matrix that is covered with a layerthat includes, e.g., streptavidin can also be utilized in certainembodiments of the invention. These particles can be used, e.g., toisolate biotin-conjugated nucleic acids and proteins. Ferrimagnetic,ferromagnetic, and superparamagnetic particles are also optionallyutilized. Magnetic glass particles and related methods that can beadapted for using in performing the methods described herein are alsodescribed in, e.g., WO 01/37291, which is incorporated by reference.

One of the most powerful and basic technologies for deriving anddetecting nucleic acids is nucleic acid amplification. In the presentinvention, amplification of nucleic acids of interest typically precedesor is concurrent with the detection of that DNA. In addition, theoligonucleotide probes described herein are also optionally amplified,e.g., following chemical synthesis or the like. In some embodiments,detectable signals are amplified, e.g., using branched nucleic acid orother signal amplification formats known in the art.

Amplification methods that are optionally utilized or adapted for usewith the oligonucleotides and methods described herein include, e.g.,various polymerase or ligase mediated amplification methods, such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA), rolling circle amplification (RCA), and/or thelike. Details regarding the use of these and other amplification methodscan be found in various articles and/or any of a variety of standardtexts, including, e.g., Berger, Sambrook, Ausubel, and PCR Protocols AGuide to Methods and Applications (Innis et al. eds) Academic Press,Inc., San Diego, Calif. (1990) (Innis), Schweitzer et al. (2001)“Combining nucleic acid amplification and detection,” Curr OpinBiotechnol. 12(1):21-27, all of which are incorporated by reference.Many available biology texts also have extended discussions regardingPCR and related amplification methods. Nucleic acid amplification isalso described in, e.g., Mullis et al., (1987) U.S. Pat. No. 4,683,202and Sooknanan and Malek (1995) Biotechnology 13:563, which are bothincorporated by reference. Improved methods of amplifying large nucleicacids by PCR are summarized in Cheng et al. (1994) Nature 369:684, whichis incorporated by reference. In certain embodiments, duplex PCR isutilized to amplify target nucleic acids. Duplex PCR amplification isdescribed further in, e.g., Gabriel et al. (2003) “Identification ofhuman remains by immobilized sequence-specific oligonucleotide probeanalysis of mtDNA hypervariable regions I and II,” Croat. Med. J.44(3)293 and La et al. (2003) “Development of a duplex PCR assay fordetection of Brachyspira hyodysenteriae and Brachyspira pilosicoli inpig feces,” J. Clin. Microbiol. 41(7):3372, which are both incorporatedby reference. Optionally, labeled primers (e.g., biotinylated primers,Scorpion primers, etc.) are utilized to amplify nucleic acids in asample, e.g., to facilitate the detection of amplicons and the like.Scorpion primers are also described in, e.g., Whitcombe et al. (1999)“Detection of PCR products using self-probing amplicons andfluorescence” Nat. Biotechnol. 17(8):804-807, which is incorporated byreference. Labeling is described further herein.

Amplicons are optionally recovered and purified from other reactioncomponents by any of a number of methods well known in the art,including electrophoresis, chromatography, precipitation, dialysis,filtration, and/or centrifugation. Aspects of nucleic acid purificationare described in, e.g., Douglas et al., DNA Chromatography, Wiley, John& Sons, Inc. (2002), and Schott, Affinity Chromatography: TemplateChromatography of Nucleic Acids and Proteins, Chromatographic ScienceSeries, #27, Marcel Dekker (1984), all of which are incorporated byreference. In certain embodiments, amplicons are not purified prior todetection. The detection of amplicons is described further below.

VII. Probe Arrays

In certain embodiments of the invention, the oligonucleotide probesdescribed herein are covalently or noncovalently attached to solidsupports which are then contacted with samples comprising amplified andlabeled nucleic acid from a subject. In other embodiments, the probes ofthe invention are provided free in solution. Essentially any substratematerial is optionally adapted for use in these aspects of theinvention. In certain embodiments, for example, substrates arefabricated from silicon, glass, or polymeric materials (e.g., glass orpolymeric microscope slides, silicon wafers, etc.). Suitable glass orpolymeric substrates, including microscope slides, are available fromvarious commercial suppliers, such as Fisher Scientific (Pittsburgh,Pa.) or the like. In some embodiments, solid supports utilized in theinvention are membranes. Suitable membrane materials are optionallyselected from, e.g. polyaramide membranes, polycarbonate membranes,porous plastic matrix membranes (e.g., POREX® Porous Plastic, etc.),porous metal matrix membranes, polyethylene membranes, poly(vinylidenedifluoride) membranes, polyamide membranes, nylon membranes, ceramicmembranes, polyester membranes, polytetrafluoroethylene (TEFLON®)membranes, woven mesh membranes, microfiltration membranes,nanofiltration membranes, ultrafiltration membranes, dialysis membranes,composite membranes, hydrophilic membranes, hydrophobic membranes,polymer-based membranes, a non-polymer-based membranes, powderedactivated carbon membranes, polypropylene membranes, glass fibermembranes, glass membranes, nitrocellulose membranes, cellulosemembranes, cellulose nitrate membranes, cellulose acetate membranes,polysulfone membranes, polyethersulfone membranes, polyolefin membranes,or the like. Many of these membranous materials are widely availablefrom various commercial suppliers, such as, P. J. Cobert Associates,Inc. (St. Louis, Mo.), Millipore Corporation (Bedford, Mass.), or thelike. Other exemplary solid supports that are optionally utilizedinclude, e.g., ceramics, metals, resins, gels, plates, beads, microbeads(e.g., magnetic microbeads, etc.), tubes (e.g., microtubes, etc.),whiskers, fibers, combs, single crystals, and self-assemblingmonolayers.

The oligonucleotide probes of the invention are directly or indirectly(e.g., via linkers, such as bovine serum albumin (BSA) or the like)attached to the supports, e.g., by any available chemical or physicalmethod. A wide variety of linking chemistries are available for linkingmolecules to a wide variety of solid supports. More specifically,nucleic acids may be attached to the solid support by covalent bindingsuch as by conjugation with a coupling agent or by non-covalent bindingsuch as electrostatic interactions, hydrogen bonds or antibody-antigencoupling, or by combinations thereof. Typical coupling agents includebiotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgGantibody F_(c) fragment, and streptavidin/protein A chimeras (Sano etal. (1991) Bio/Technology 9:1378, which is incorporated by reference),or derivatives or combinations of these agents. Nucleic acids may beattached to the solid support by a photocleavable bond, an electrostaticbond, a disulfide bond, a peptide bond, a diester bond or a combinationof these bonds. Nucleic acids are also optionally attached to solidsupports by a selectively releasable bond such as 4,4′-dimethoxytritylor its derivative. Derivatives which have been found to be usefulinclude 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]hydroxymethyl-benzoic acid,N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid,and salts of these acids.

As referred to above, oligonucleotide probes are optionally attached tosolid supports via linkers between the nucleic acids and the solidsupport. Useful linkers include a coupling agent, as described above forbinding to other or additional coupling partners, or to render theattachment to the solid support cleavable.

Cleavable attachments can be created by attaching cleavable chemicalmoieties between the probes and the solid support including, e.g., anoligopeptide, oligonucleotide, oligopolyamide, oligoacrylamide,oligoethylene glycerol, alkyl chains of between about 6 to 20 carbonatoms, and combinations thereof. These moieties may be cleaved with,e.g., added chemical agents, electromagnetic radiation, or enzymes.Exemplary attachments cleavable by enzymes include peptide bonds whichcan be cleaved by proteases, and phosphodiester bonds which can becleaved by nucleases.

Chemical agents such as β-mercaptoethanol, dithiothreitol (DTT) andother reducing agents cleave disulfide bonds. Other agents which may beuseful include oxidizing agents, hydrating agents and other selectivelyactive compounds. Electromagnetic radiation such as ultraviolet,infrared and visible light cleave photocleavable bonds. Attachments mayalso be reversible, e.g., using heat or enzymatic treatment, orreversible chemical or magnetic attachments. Release and reattachmentcan be performed using, e.g., magnetic or electrical fields.

Array based hybridization is particularly suitable for detecting N.gonorrhoeae and/or C. trachomatis nucleic acids, as it can be used todetect the presence of many amplicons simultaneously. A number of arraysystems have been described and can be adapted for use with the presentinvention, including those available from commercial suppliers such asAffymetrix, Inc. (Santa Clara, Calif., USA) and the like. Aspects ofarray construction and use are also described in, e.g., Sapolsky et al.(1999) “High-throughput polymorphism screening and genotyping withhigh-density oligonucleotide arrays.” Genetic Analysis: BiomolecularEngineering 14:187-192; Lockhart (1998) “Mutant yeast on drugs” NatureMedicine 4:1235-1236; Fodor (1997) “Genes, Chips and the Human Genome.”FASEB Journal 11:A879; Fodor (1997) “Massively Parallel Genomics”Science 277: 393-395; and Chee et al. (1996) “Accessing GeneticInformation with High-Density DNA Arrays” Science 274:610-614, all ofwhich are incorporated by reference.

Other probes and primers for detecting N. gonorrhoeae and/or C.trachomatis nucleic acids, which are optionally utilized in addition tothe probes and primer described above to perform the methods and otheraspects of the invention, are described in, e.g., U.S. Pat. No.5,550,040 to Purohit et al., and U.S. Pat. No. 6,090,557 to Weiss, whichare both incorporated by reference.

VIII. Nucleic Acid Hybridization

Hybridization of oligonucleotide probes to their target N. gonorrhoeaeand/or C. trachomatis nucleic acids can be accomplished by choosing theappropriate hybridization conditions. The stability of the probe:targetnucleic acid hybrid is typically selected to be compatible with theassay and washing conditions so that stable, detectable hybrids formonly between the probes and target N. gonorrhoeae and/or C. trachomatisnucleic acids. Manipulation of one or more of the different assayparameters determines the exact sensitivity and specificity of aparticular hybridization assay.

More specifically, hybridization between complementary bases of DNA,RNA, PNA, or combinations of DNA, RNA and PNA, occurs under a widevariety of conditions that vary in temperature, salt concentration,electrostatic strength, buffer composition, and the like. Examples ofthese conditions and methods for applying them are described in, e.g.,Tijssen (1993), supra, and Hames and Higgins, supra. Hybridizationgenerally takes place between about 0° C. and about 70° C., for periodsof from about one minute to about one hour, depending on the nature ofthe sequence to be hybridized and its length. However, it is recognizedthat hybridizations can occur in seconds or hours, depending on theconditions of the reaction. To illustrate, typical hybridizationconditions for a mixture of two 20-mers is to bring the mixture to 68°C., followed by cooling to room temperature (22° C.) for five minutes orat very low temperatures such as 2° C. in 2 microliters. Hybridizationbetween nucleic acids may be facilitated using buffers such as Tris-EDTA(TE), Tris-HCl and HEPES, salt solutions (e.g. NaCl, KCl, CaCl₂), orother aqueous solutions, reagents and chemicals. Examples of thesereagents include single-stranded binding proteins such as Rec A protein,T4 gene 32 protein, E. coli single-stranded binding protein and major orminor nucleic acid groove binding proteins. Other examples of suchreagents and chemicals include divalent ions, polyvalent ions andintercalating substances such as ethidium bromide, actinomycin D,psoralen, and angelicin. An exemplary hybridization procedure of use inthe present invention follows similar conditions as specified in theCOBAS AMPLICOR® Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NC)Test protocol (Roche Diagnostics Corporation, Indianapolis, Ind.).

IX. Detection and Probe Variations

As referred to above, amplified target N. gonorrhoeae and/or C.trachomatis nucleic acid in the samples utilized in the methods of theinvention is optionally labeled to permit detection of oligonucleotideprobe-target hybridization duplexes. In general, a label can be anymoiety that can be attached, e.g., to a primer utilized foramplification and provide a detectable signal (e.g., a quantifiablesignal). Labels may be attached to a primer directly or indirectly by avariety of techniques known in the art. Depending on the type of labelused, the label can be attached to a terminal (5′ or 3′ end of theprimer) or a non-terminal nucleotide, and can be attached indirectlythrough linkers or spacer arms of various sizes and compositions. Usingcommercially available phosphoramidite reagents, one can produceoligomers containing functional groups (e.g., thiols or primary amines)at either the 5′ or 3′ terminus via an appropriately protectedphosphoramidite, and can label such oligonucleotides using protocolsdescribed in, for example, PCR Protocols: A Guide to Methods andApplications (Innis et al, eds. Academic Press, Inc. (1990)). In oneembodiment, the label consists of a biotin molecule covalently bound tothe primer at the 5′ end. The term “biotinylated primer” refers to aprimer with one or more biotin molecules bound either directly to theprimer or indirectly through intervening linker molecules.

To further illustrate, detection of oligonucleotide probe-targethybridization duplexes is optionally by a chemiluminescent assay using aluminol-based reagent as described in, e.g., Whitehead, et al. (1983)Nature 30(5):158, which is incorporated by reference, and availablecommercially. Following hybridization of the probe with the labeledtarget DNA, the biotin molecule attached to the target DNA isconjugated, e.g., to streptavidin-horseradish peroxidase (SA-HRP).Alternatively, the target DNA can be labeled with horseradish peroxidasedirectly, thereby eliminating the separate conjugation step. In eithercase, subsequent oxidation of luminol by the horseradish peroxidaseenzyme results in the emission of photons, which is then detected, e.g.,on standard autoradiography film. The intensity of the signal is afunction of DNA quantity. A series of DNA standards containing knownamounts of DNA are typically assayed along with one or more unknownsamples. The signal intensities of the known DNA standards allow anempirical determination of the functional relationship between signalintensity and DNA quantity, which enables the quantitation of theunknown samples. Many other methods of detection are also optionallyutilized to perform the methods of the invention and are referred to inthe references cited herein and/or generally known in the art.

Any available method for detecting N. gonorrhoeae and/or C. trachomatisamplicons can be used in the present invention. Common approachesinclude real time amplification detection with molecular beacons or5′-nuclease probes, detection of intercalating dyes, detection of labelsincorporated into the amplification probes or the amplified nucleicacids themselves, e.g., following electrophoretic separation of theamplification products from unincorporated label), hybridization basedassays (e.g., array based assays) and/or detection of secondary reagentsthat bind to the nucleic acids. For example, NG and/or CT is detectedusing the oligonucleotides described herein in an AMPLICOR® testingformat in certain embodiments of the invention.

To further illustrate, a molecular beacon or a 5′-nuclease probe isoptionally designed to include a oligonucleotide probe of the invention(i.e., is selected from SEQ ID NOS: 3-27) or complements thereto), whichmolecular beacon or 5′-nuclease probe can be used to detect N.gonorrhoeae and/or C. trachomatis amplicons. Molecular beacons or5′-nuclease probes are described further below. Details on these generalapproaches are found in the references cited herein, e.g., Sambrook andAusubel. Additional labeling strategies for labeling nucleic acids andcorresponding detection strategies can be found, e.g., in Haugland(2003) Handbook of Fluorescent Probes and Research Chemicals NinthEdition by Molecular Probes, Inc. (Eugene, Oreg.), which is incorporatedby reference.

Molecular beacons (MBs) are oligonucleotides designed for real timedetection and quantification of target nucleic acids (e.g., target N.gonorrhoeae and/or C. trachomatis amplicons). The 5′ and 3′ termini ofMBs collectively comprise a pair of moieties which confers thedetectable properties of the MB. One of the termini is attached to afluorophore and the other is attached to a quencher molecule capable ofquenching a fluorescent emission of the fluorophore. For example, oneexample fluorophore-quencher pair can use a fluorophore such as EDANS orfluorescein, e.g., on the 5′-end and a quencher such as Dabcyl, e.g., onthe 3′-end. When the MB is present free in solution, i.e., nothybridized to a second nucleic acid, the stem of the MB is stabilized bycomplementary base pairing. This self-complementary pairing results in a“hairpin loop” structure for the MB in which the fluorophore and thequenching moieties are proximal to one another. In this confirmation,the fluorescent moiety is quenched by the fluorophore. The loop of themolecular beacon typically comprises an oligonucleotide probe describedherein (i.e., is selected from SEQ ID NOS: 3-27 and 37-60 or complementsthereto) and is accordingly complementary to a sequence to be detectedin the target N. gonorrhoeae and/or C. trachomatis nucleic acid, suchthat hybridization of the loop to its complementary sequence in thetarget forces disassociation of the stem, thereby distancing thefluorophore and quencher from each other. This results in unquenching ofthe fluorophore, causing an increase in fluorescence of the MB.

Details regarding standard methods of making and using MBs are wellestablished in the literature and MBs are available from a number ofcommercial reagent sources. Further details regarding methods of MBmanufacture and use are found, e.g., in Leone et al. (1995) “Molecularbeacon probes combined with amplification by NASBA enable homogenousreal-time detection of RNA,” Nucleic Acids Res. 26:2150-2155; Hsuih etal. (1997) “Novel, ligation-dependent PCR assay for detection ofhepatitis C in serum” J Clin Microbiol 34:501-507; Kostrikis et al.(1998) “Molecular beacons: spectral genotyping of human alleles” Science279:1228-1229; Sokol et al. (1998) “Real time detection of DNA:RNAhybridization in living cells” Proc. Natl. Acad. Sci. U.S.A.95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons forallele discrimination” Nature Biotechnology 16:49-53; Fang et al. (1999)“Designing a novel molecular beacon for surface-immobilized DNAhybridization studies” J. Am. Chem. Soc. 121:2921-2922; and Marras etal. (1999) “Multiplex detection of single-nucleotide variation usingmolecular beacons” Genet. Anal. Biomol. Eng. 14:151-156, all of whichare incorporated by reference. Aspects of MB construction and use arealso found in patent literature, such as U.S. Pat. No. 5,925,517 (Jul.20, 1999) to Tyagi et al. entitled “Detectably labeled dual conformationoligonucleotide probes, assays and kits;” U.S. Pat. No. 6,150,097 toTyagi et al (Nov. 21, 2000) entitled “Nucleic acid detection probeshaving non-FRET fluorescence quenching and kits and assays includingsuch probes” and U.S. Pat. No. 6,037,130 to Tyagi et al (Mar. 14, 2000),entitled “Wavelength-shifting probes and primers and their use in assaysand kits,” all of which are incorporated by reference.

MB components (e.g., oligos, including those labeled with fluorophoresor quenchers) can be synthesized using conventional methods. Some ofthese methods are described further above. For example, oligonucleotidesor peptide nucleic acids (PNAs) can be synthesized on commerciallyavailable automated oligonucleotide/PNA synthesis machines usingstandard methods. Labels can be attached to the oligonucleotides or PNAseither during automated synthesis or by post-synthetic reactions whichhave been described before see, e.g., Tyagi and Kramer (1996), supra.Aspects relating to the synthesis of functionalized oligonucleotides canalso be found in Nelson, et al. (1989) “Bifunctional OligonucleotideProbes Synthesized Using A Novel CPG Support Are Able To Detect SingleBase Pair Mutations” Nucleic Acids Res. 17:7187-7194, which isincorporated by reference. Labels/quenchers can be introduced to theoligonucleotides or PNAs, e.g., by using a controlled-pore glass columnto introduce, e.g., the quencher (e.g., a4-dimethylaminoazobenzene-4′-sulfonyl moiety (DABSYL). For example, thequencher can be added at the 3′ end of oligonucleotides during automatedsynthesis; a succinimidyl ester of 4-(4′-dimethylaminophenylazo)benzoicacid (DABCYL) can be used when the site of attachment is a primary aminogroup; and 4-dimethylaminophenylazophenyl-4′-maleimide (DABMI) can beused when the site of attachment is a sulphydryl group. Similarly,fluorescein can be introduced in the oligonucleotides, either using afluorescein phosphoramadite that replaces a nucleoside with fluorescein,or by using a fluorescein dT phosphoramadite that introduces afluorescein moiety at a thymidine ring via a linker. To link afluorescein moiety to a terminal location, iodoacetoamidofluorescein canbe coupled to a sulphydryl group, Tetrachlorofluorescein (TET) can beintroduced during automated synthesis using a 5′-tetrachloro-fluoresceinphosphoramadite. Other reactive fluorophore derivatives and theirrespective sites of attachment include the succinimidyl ester of5-carboxyrhodamine-6G (RHD) coupled to an amino group; an iodoacetamideof tetramethylrhodamine coupled to a sulphydryl group; an isothiocyanateof tetramethylrhodamine coupled to an amino group; or a sulfonylchlorideof Texas red coupled to a sulphydryl group. During the synthesis ofthese labeled components, conjugated oligonucleotides or PNAs can bepurified, if desired, e.g., by high pressure liquid chromatography orother methods.

A variety of commercial suppliers produce standard and custom molecularbeacons, including Cruachem (cruachem.com), Oswel Research Products Ltd.(UK; oswel.com), Research Genetics (a division of Invitrogen, HuntsvilleAla. (resgen.com)), the Midland Certified Reagent Company (Midland, Tex.mcrc.com) and Gorilla Genomics, LLC (Alameda, Calif.). A variety ofkits, which utilize molecular beacons are also commercially available,such as the Sentinel™ Molecular Beacon Allelic Discrimination Kits fromStratagene (La Jolla, Calif.) and various kits from Eurogentec SA(Belgium, eurogentec.com) and Isogen Bioscience BV (The Netherlands,isogen.com).

In certain embodiments, a real time PCR assay system that includes oneor more 5′-nuclease probes is used for detecting amplified N.gonorrhoeae and/or C. trachomatis nucleic acids. These systems operateby using the endogenous nuclease activity of certain polymerases tocleave a quencher or label free from an oligonucleotide of the inventionthat comprises the quencher and label, resulting in unquenching of thelabel. The polymerase only cleaves the quencher or label upon initiationof replication, i.e., when the oligonucleotide is bound to the templateand the polymerase extends the primer. Thus, an appropriately labeledoligonucleotide probe and polymerase comprising the appropriate nucleaseactivity can be used to detect an N. gonorrhoeae and/or C. trachomatisnucleic acid of interest. Real time PCR product analysis by, e.g., FRETor the like (and related real time reverse-transcription PCR) provides awell-known technique for real time PCR monitoring that has been used ina variety of contexts, which can be adapted for use with the probes andmethods described herein (see, Laurendeau et al. (1999) “TaqManPCR-based gene dosage assay for predictive testing in individuals from acancer family with INK4 locus haploinsufficiency” Clin Chem 45(7):982-6;Laurendeau et al. (1999) “Quantitation of MYC gene expression insporadic breast tumors with a real-time reverse transcription-PCR assay”Clin Chem 59(12):2759-65; and Kreuzer et al. (1999) “LightCyclertechnology for the quantitation of bcr/abl fusion transcripts” CancerResearch 59(13):3171-4, all of which are incorporated by reference).

X. Systems

The invention also provides a system for detecting N. gonorrhoeae and/orC. trachomatis in a sample. The system includes one or more nucleic aciddetection reagents as described herein (e.g., probe nucleic acids,sequence specific antibodies, etc.). In certain embodiments, the nucleicacid detection reagents are arrayed on a solid support, whereas inothers, they are provided in one or more containers, e.g., for assaysperformed in solution. The system also includes at least one detector(e.g., a spectrometer, etc.) that detects binding between nucleic acidsand/or amplicons thereof from the sample and the nucleic acid detectionreagent. Other detectors are described further below. In addition, thesystem also includes at least one controller operably connected to thedetector. The controller includes one or more instructions sets thatcorrelate the binding detected by the detector with a presence ofNeisseria gonorrhoeae and/or C. trachomatis in the sample.

In some embodiments, at least one container the nucleic acid detectionreagent. In these embodiments, the system optionally further includes atleast one thermal modulator operably connected to the container tomodulate temperature in the container, and/or at least one fluidtransfer component (e.g., an automated pipettor, etc.) that transfersfluid to and/or from the container, e.g., for performing one or morenucleic acid amplification techniques in the container, etc.

Exemplary commercially available systems that are optionally utilized todetect N. gonorrhoeae and/or C. trachomatis nucleic acids using thenucleic acid detection reagents described herein (e.g., oligonucleotideprobes comprising sequences selected from the group consisting of: SEQID NOS: 3-27 and 37-60 or complements thereto, sequence specificantibodies, etc.) include, e.g., a COBAS AMPLICOR® Analyzer, which isavailable from Roche Diagnostics Corporation (Indianapolis, Ind.), aLININEX 100™ system, which is available from the Luminex Corporation(Austin, Tex.), an ABI PRISM® Sequence Detection System, which isavailable from Applied Biosystems (Foster City, Calif.), and the like.

The invention further provides a computer or computer readable mediumthat includes a data set that comprises a plurality of character stringsthat correspond to a plurality of sequences that correspond tosubsequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, asubstantially identical variant thereof in which the variant has atleast 90% sequence identity to one of SEQ ID NOS: 1 or 2 or 36, or acomplement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 36, or the variant.Typically, at least one of the character strings corresponds to asequence selected from the group consisting of: SEQ ID NOS: 3-27 and37-60 or complements thereof. Typically, the computer or computerreadable medium further includes an automatic synthesizer coupled to anoutput of the computer or computer readable medium. The automaticsynthesizer accepts instructions from the computer or computer readablemedium, which instructions direct synthesis of, e.g., one or more probenucleic acids that correspond to one or more character strings in thedata set. Exemplary systems and system components are described furtherbelow.

Detectors are structured to detect detectable signals produced, e.g., inor proximal to another component of the system (e.g., in container, on asolid support, etc.). Suitable signal detectors that are optionallyutilized, or adapted for use, in these systems detect, e.g.,fluorescence, phosphorescence, radioactivity, absorbance, refractiveindex, luminescence, or the like. Detectors optionally monitor one or aplurality of signals from upstream and/or downstream of the performanceof, e.g., a given assay step. For example, the detector optionallymonitors a plurality of optical signals, which correspond in position to“real time” results. Example detectors or sensors includephotomultiplier tubes, CCD arrays, optical sensors, temperature sensors,pressure sensors, pH sensors, conductivity sensors, scanning detectors,or the like. Each of these as well as other types of sensors isoptionally readily incorporated into the systems described herein.Optionally, the systems of the present invention include multipledetectors.

More specific exemplary detectors that are optionally utilized in thesesystems include, e.g., a resonance light scattering detector, anemission spectroscope, a fluorescence spectroscope, a phosphorescencespectroscope, a luminescence spectroscope, a spectrophotometer, aphotometer, and the like. Various synthetic components are alsoutilized, or adapted for, use in the systems of the invention including,e.g., automated nucleic acid synthesizers, e.g., for synthesizing theoligonucleotides probes described herein. Detectors and syntheticcomponents that are optionally included in the systems of the inventionare described further in, e.g., Skoog et al., Principles of InstrumentalAnalysis, 5^(th) Ed., Harcourt Brace College Publishers (1998) andCurrell, Analytical Instrumentation: Performance Characteristics andQuality, John Wiley & Sons, Inc. (2000), both of which are incorporatedby reference.

The systems of the invention also typically include controllers that areoperably connected to one or more components (e.g., detectors, syntheticcomponents, thermal modulator, fluid transfer components, etc.) of thesystem to control operation of the components. More specifically,controllers are generally included either as separate or integral systemcomponents that are utilized, e.g., to receive data from detectors, toeffect and/or regulate temperature in the containers, to effect and/orregulate fluid flow to or from selected containers, or the like.Controllers and/or other system components is/are optionally coupled toan appropriately programmed processor, computer, digital device, orother information appliance (e.g., including an analog to digital ordigital to analog converter as needed), which functions to instruct theoperation of these instruments in accordance with preprogrammed or userinput instructions, receive data and information from these instruments,and interpret, manipulate and report this information to the user.Suitable controllers are generally known in the art and are availablefrom various commercial sources.

Any controller or computer optionally includes a monitor, which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display, etc.), or others.Computer circuitry is often placed in a box, which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser. These components are illustrated further below.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set of parameterfields, e.g., in a Gut, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of one or more controllers to carry out thedesired operation. The computer then receives the data from, e.g.,sensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as controlling fluid flow regulators in responseto fluid weight data received from weight scales or the like.

The computer can be, e.g., a PC (Intel x86 or Pentium chip-compatibleDOS™, OS2™, WINDOWS™, WINDOWS NT™, WINDOWS95™, WINDOWS98™, WINDOWS2000™,WINDOWS XP™, LINUX-based machine, a MACINTOSH™, Power PC, or aUNIX-based (e.g., SUN™ work station) machine) or other commoncommercially available computer which is known to one of skill. Standarddesktop applications such as word processing software (e.g., MicrosoftWord™ or Corel WordPerfect™) and database software (e.g., spreadsheetsoftware such as Microsoft Excel™, Corel Quattro Pro™, or databaseprograms such as Microsoft Access™ or Paradox™) can be adapted to thepresent invention. Software for performing, e.g., controllingtemperature modulators and fluid flow regulators is optionallyconstructed by one of skill using a standard programming language suchas Visual basic, Fortran, Basic, Java, or the like.

FIGS. 2 and 3 are schematics showing representative example systems thatinclude logic devices in which various aspects of the present inventionmay be embodied. As will be understood by practitioners in the art fromthe teachings provided herein, the invention is optionally implementedin hardware and/or software. In some embodiments, different aspects ofthe invention are implemented in either client-side logic or server-sidelogic. As will be understood in the art, the invention or componentsthereof may be embodied in a media program component (e.g., a fixedmedia component) containing logic instructions and/or data that, whenloaded into an appropriately configured computing device, cause thatdevice to perform according to the invention. As will also be understoodin the art, a fixed media containing logic instructions may be deliveredto a viewer on a fixed media for physically loading into a viewer'scomputer or a fixed media containing logic instructions may reside on aremote server that a viewer accesses through a communication medium inorder to download a program component.

In particular, FIG. 2 schematically illustrate computer 200 to whichdetector 202 and fluid transfer component 204 are operably connected.Optionally, detector 202 and/or fluid transfer component 204 is operablyconnected to computer 200 via a server (not shown in FIG. 2). Duringoperation, fluid transfer component 204 typically transfers fluids, suchas sample aliquots comprising labeled N. gonorrhoeae and/or C.trachomatis amplicons to nucleic acid detection reagent array 206, e.g.,comprising oligonucleotide probes, sequence specific antibodies, etc.,as described herein, arrayed thereon. Thereafter, detector 202 typicallydetects detectable signals (e.g., fluorescent emissions, etc.) producedby labeled amplicons that hybridize with probes attached to nucleic aciddetection reagent array 206 after one or more washing steps areperformed to wash away non-hybridized nucleic acids from nucleic aciddetection reagent array 206 using fluid transfer component 204. Asadditionally shown, thermal modulator 208 is also operably connected tocomputer 200. Prior to performing a hybridization assay, target N.gonorrhoeae and/or C. trachomatis nucleic acids can be amplified usinglabeled primer nucleic acids (e.g., primers comprising sequencesselected from SEQ ID NOS: 3-27 and 37-60). The amplicons of theseamplification reactions are then typically transferred to nucleic aciddetection reagent array 206 using fluid transfer component 204, asdescribed above, to perform the binding assay. In some embodiments,binding assays are performed concurrently with N. gonorrhoeae and/or C.trachomatis nucleic acid amplification in thermal modulator 208 using,e.g., molecular beacons, 5′-nuclease probes, or the like that comprisesequences selected from SEQ ID NOS: 3-27 and 37-60. In theseembodiments, detector 202 detects detectable signals produced as theamplification reactions are performed using thermal modulator 208.

FIG. 3 schematically shows information appliance or digital device 300that may be understood as a logical apparatus that can read instructionsfrom media 302 and/or network port 304, which can optionally beconnected to server 306 having fixed media 308. Digital device 300 canthereafter use those instructions to direct server or client logic, asunderstood in the art, to embody aspects of the invention. One type oflogical apparatus that may embody the invention is a computer system asillustrated in 300, containing CPU 310, optional input devices 312 and314, disk drives 316 and optional monitor 318. Fixed media 302, or fixedmedia 308 over port 304, may be used to program such a system and mayrepresent a disk-type optical or magnetic media, magnetic tape, solidstate dynamic or static memory, or the like. In specific embodiments,the invention may be embodied in whole or in part as software recordedon this fixed media. Communication port 304 may also be used toinitially receive instructions that are used to program such a systemand may represent any type of communication connection. Optionally, theinvention is embodied in whole or in part within the circuitry of anapplication specific integrated circuit (ACIS) or a programmable logicdevice (PLD). In such a case, the invention may be embodied in acomputer understandable descriptor language, which may be used to createan ASIC, or PLD.

FIG. 3 also includes automatic synthesizer 320, which is operablyconnected to digital device 300 via server 306. Optionally, automaticsynthesizer 320 is directly connected to digital device 300. Duringoperation, automatic synthesizer 320 typically receives instructions tosynthesize one or more primers or probes that comprise a sequenceselected from the group consisting of: SEQ ID NOS: 3-27 and 37-60 orcomplements thereto, which are included in a data set comprised by,e.g., digital device 300 and/or a computer readable medium, such asfixed media 302 and/or 308.

XI. Kits

The nucleic acid detection reagents employed in the methods of thepresent invention are optionally packaged into kits. As describedherein, the nucleic acid detection reagents of the invention detectablybind to a nucleic acid with a sequence consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 36, a substantially identical variant thereof inwhich the variant has at least 90% sequence identity to one of SEQ IDNOS: 1 or 2 or 36, or a complement of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 36, or the variant. In addition, the kits may also include suitablypackaged reagents and materials needed for DNA immobilization,hybridization, and/or detection, such solid supports, buffers, enzymes,and DNA standards, as well as instructions for conducting the assay.Optionally, the nucleic acid detection reagents (e.g., oligonucleotideprobes, sequence specific antibodies, etc.) of the invention areprovided already attached or otherwise immobilized on solid supports. Asanother option, nucleic acid detection reagents are provided free insolution in containers, e.g., for performing the detection methods ofthe invention in the solution phase. In some of these embodiments,nucleic acid detection reagents of the kits comprise labels and/orquencher moieties, such as when molecular beacons, 5′-nuclease probes,or the like comprise sequences selected from SEQ ID NOS: 3-27 and 37-60.In certain embodiments, kits further include labeled primers foramplifying target N. gonorrhoeae and/or C. trachomatis sequences in asample.

The kit also includes one or more of: a set of instructions forcontacting the nucleic acid detection reagents with nucleic acids from asample or amplicons thereof and detecting binding between the nucleicacid detection reagents and N. gonorrhoeae and/or C. trachomatis nucleicacids, if any, or at least one container for packaging the nucleic aciddetection reagents and the set of instructions. Exemplary solid supportsinclude in the kits of the invention are optionally selected from, e.g.,a plate, a microwell plate, a bead, a microbead, a tube (e.g., amicrotube, etc.), a fiber, a whisker, a comb, a hybridization chip, amembrane, a single crystal, a ceramic layer, a self-assemblingmonolayer, or the like.

In some embodiments, the kit further includes at least one primernucleic acid that is at least partially complementary to at least onesegment of an N. gonorrhoeae nucleic acid, e.g., for amplifying asegment of the N. gonorrhoeae nucleic acid. In certain embodiments, thekit also includes one or more primers for amplifying one or moresegments of a C. trachomatis nucleic acid. In these embodiments, the kittypically further includes a set of instructions for amplifying one ormore subsequences of those nucleic acids with the primer nucleic acids,at least one nucleotide incorporating biocatalyst, and one or morenucleotides. In certain embodiments, the primer nucleic acids compriseat least one label (e.g., a fluorescent dye, a radioisotope, etc.).Suitable labels are described further herein. For example, the primernucleic acid is optionally conjugated with biotin or a biotinderivative. In these embodiments, the kit typically further includes anenzyme conjugated with avidin or an avidin derivative, or streptavidinor a streptavidin derivative, e.g., for effecting the detection ofbinding between the nucleic acid detection reagents of the invention andtarget nucleic acids. In these embodiments, the kit generally furtherincludes at least one nucleotide incorporating biocatalyst (e.g., apolymerase, a ligase, or the like). In these embodiments, the kittypically also further comprising one or more nucleotides, e.g., for usein amplifying the target nucleic acids. Optionally, at least one of thenucleotides comprises a label. In some of these embodiments, the kitsfurther include at least one pyrophosphatase (e.g., a thermostablepyrophosphatase), e.g., for use in minimizing pyrophosphorolysis, uracilN-glycosylase (UNG) (e.g., a thermostable UNG), e.g., for use inapplications where protection against carry-over contamination isdesirable.

XII. EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and are not intended to limit the scopeof the claimed invention. It is also understood that variousmodifications or changes in light the examples and embodiments describedherein will be suggested to persons skilled in the art and are to beincluded within the spirit and purview of this application and scope ofthe appended claims.

Example I Detection of N. gonorrhoeae Via Neisseria gonorrhoeae DirectRepeat 9

Selection and Synthesis of Neisseria gonorrhoeae Direct Repeat 9Specific Oligonucleotide Primers for PCR Analysis

The Neisseria gonorrhoeae Direct Repeat 9 (NGDR9) previously wasidentified as a two-copy DNA sequence in the Neisseria gonorrhoeaegenome. The sequence of NGDR9 was obtained from the Los Alamos NationalLaboratory Sexually Transmitted Diseases database via the world wide webat stdgen.lanl.gov/stdgen/bacteria/ngon/. The entire 806 base pair NGDR9sequence lacks substantial identity with any sequence in the Neisseriameningitidis genome, but has 36.85% identity with gaps (44.4% identitywithout gaps) to Brucella suis 1330 chromosome I section 155 (GenBank®accession number AE014469). FIG. 4 depicts a Clustal W alignment of theNGDR9 sequence (SEQ ID NO: 1) with a portion of this Brucella sequence(SEQ ID NO:34). The NGDR9 sequence was scanned for regions of minimalsequence identity with B. suis and upstream and downstreamoligonucleotide primers (NG519 (5′-CTCTCAATGCCCAATCATAAAGC-3′ (SEQ IDNO:5)) and a complement to NG514 (i.e., 5′-GATAAAGCAGACGAAGCGGATAC-3′(SEQ ID NO: 24)), respectively spanning a 190 base pair region of NGDR9were synthesized. The deoxycytidylate units at the 3′ ends of both ofthese primers had been modified to include t-butyl benzyl groups asdescribed in, e.g., U.S. Pat. No. 6,001,611, entitled “MODIFIED NUCLEICACID AMPLIFICATION PRIMERS,” issued Dec. 14, 1999 to Will, which isincorporated by reference. The positions of NG519 and NG514 in NGDR9 areunderlined in FIG. 4.

The positions of other exemplary upstream and downstream oligonucleotideprimer pairs are also underlined in FIG. 4. In particular, DK101(5′-GTTTGGCGGCAAGCATCT-3′ (SEQ ID NO:7)) and DK102(5′-AAATGGGATGCTGTCGTCAA-3′ (SEQ ID NO:8)) are shown. The primer pairDK101 and a complement to DK102 (i.e., 5′-TTGACGACAGCATCCCATTT-3′ (SEQID NO. 25)) is designed to amplify a 416 base pair region of NGDR9.Primers corresponding to DK101 and the complement to DK 102, whichfurther included 5′-end restriction site linkers were also synthesized,namely, HINDDK101 (5′-GGCAAGCTTGTTTGGCCGGCAAGCATCT-3′ (SEQ ID NO:9);HindIII restriction site underlined) and BAMDK102(5′-GGCGGATCCTTGACGACAGCATCCCATTT-3′ (SEQ ID NO:10); BamHI restrictionsite underlined). A photograph of an agarose gel that shows thedetection of N. gonorrhoeae using this pair of primers is providedbelow. DK103 (5′-AAACGCAATCTTCAAACACCTCA-3′ (SEQ ID NO:1)) and DK104(5′-TTTGACGGCCTCACGCATAA-3′ (SEQ ID NO: 12)) are also shown underlinedin FIG. 4. The primer pair DK103 and a complement to DK104 (i.e.,5′-TTATGCGTGAGGCCGTCAAA-3′ (SEQ ID NO: 26)) is designed to amplify a 384base pair region of NGDR9.

Neisserial Genomic DNA Purification

Extraction of genomic DNA from various neisserial strains was performedby using the PureGene® DNA Purification system (Gentra Systems,Minneapolis Minn.). Bacterial cells were grown for 48 hours on Chocolateagar (Hardy Diagnostics, Santa Maria, Calif.) at 37° C. in 5% CO₂. Thecells were scraped from the agar surface, re-suspended in PBS andcentrifuged at 13,000-16,000×g for 5 seconds to pellet the cells. Thesupernatant was removed by aspiration, leaving behind 10-20 μl residualliquid. The samples were vortexed vigorously to re-suspend the pellet inthe residual liquid. DNA extraction was carried out as instructed by themanufacturers. Briefly, 300 μl cell lysis solution was added to there-suspended cells and pipetted up and down to lyse the cells. Followingaddition of 1.5 μl of RNAse A solution to the cell lysate, the sampleswere mixed by inverting the tubes 25 times and incubated for 5 minutesat 37° C. The samples were cooled to room temperature by being placed onice for 1 minute. A 100 μl volume of Protein Precipitation Solution wasadded to the RNAse-treated cell lysate and the samples were mixed byvortexing vigorously at high speed for 20 seconds. The protein debriswas precipitated by centrifugation at 13,000-16,000×g for 1 minute. Thesupernate containing the DNA samples was transferred into clean 1.5 mlmicro centrifuge tubes, each containing 300 μl of 100% isopropanol. Thesamples were mixed by gently inverting the tubes 50 times and were thencentrifuged at 13,000-16,000×g for 1 minute. The supernate was pouredoff and the pellet washed with 300 μl 70% ethanol. The tubes werecentrifuged again at 13,000-16,000×g for 1 minute. After removal of thesupernate, the tubes were drained by inversion and the DNA pellets werere-suspended in 50 μl of Hydration Solution. Genomic DNA was quantitatedusing the PicoGreen dsDNA Quantitation Reagents (Molecular Probes,Eugene, Oreg.) and the re-suspended DNA was stored at −20° C. untilused.

Amplification of Segments of NGDR9

Separate PCR reactions were performed using genomic DNA isolated fromthe various neisserial species as templates and the pair of primers,NG519 and the complement to NG514 (described above). PCRs were performedin volumes of 100 μl containing 50 mM Tricine (pH 8.3), 80 mM K(OAc)₂(pH 7.5), 2 mM Mn(OAc)₂ (pH 6.5), 50 μM dATP, 50 μM dGTP, 50 μM dCTP,100 μM dUTP, 20 U of ZO5 DNA polymerase (Roche Molecular Systems,Alameda, Calif.), 5U AmpErase® UNG (Uracil-N-Glycosylase), 0.5 μM ofeach primer and 1 ng/μl ethidium bromide. Genomic DNA was added astemplate at 10 genomic equivalents per reaction for Neisseriagonorrhoeae and 10⁶ genomic equivalents per reaction for Neisseriameningitidis and other neisserial strains. Reactions were performed for60 cycles of denaturation at 95° C. for 15 seconds, annealing at 58° C.for 20 seconds, and a final extension at 72° C. for 5 minutes using aCOBAS TaqMan® PCR System (Roche Molecular Systems, Alameda, Calif.).

Separate PCR reactions were also performed using genomic DNA isolatedfrom various neisserial species as templates and the pair of primers,HINDDK101 and BAMDK102 (described above), using a procedure similar tothat described above used to amplify the 190 base pair segment of NGDR9.

Analytical Agarose Gel Electrophoresis of PCR Products

The PCR products were prepared for gel electrophoresis analysis byadding 20 μl of the DNA samples to 8 μl of 10× gel loading buffer(0.025% bromophenol blue dye, 100 mM EDTA, and 30% sucrose). The sampleswere then loaded into lanes of a horizontally submerged gel containing a3.0% (w/v) Nusieve, 0.5% (w/v) agarose gel and 0.5 μg/ml ethidiumbromide in 1×TB buffer (0.089M Tris, 0.09M Boric Acid, 2 mM EDTA, pH8.0). The electrophoresis running buffer was 1×TB buffer containing 0.5μg/ml ethidium bromide. The gel was run at 95-100 V for 1 hour, thenremoved and visualized on a long wavelength UV transilluminator. Theparticular gels were examined for the presence or absence of PCRamplification products at the expected sizes of 190 or 416 bp.

FIGS. 5 A and B, and 6 A and B are photographs of agarose gels that showthe detection of the 190 base pair segment of NGDR9, whereas FIG. 7 is aphotograph of an agarose gel that shows the detection of the 416 basepair segment of NGDR9.

Example II Assays Illustrating the Selective Detection of N. gonorrhoeae

This example provides lists of organisms that were analyzed in assaysthat included the use of primer nucleic acids NG519 (having a sequencecorresponding to SEQ ID NO: 5) and NG514R (having a sequencecorresponding to SEQ ID NO: 24) and a 5′-nuclease probe (having asequence corresponding to SEQ ID NO: 18) alone or in combination with C.trachomatis primers. In particular, the inclusivity (i.e., a measure ofthe ability to detect the target organism, N. gonorrhoeae in samples) ofthese assays is illustrated in Table XX.

TABLE IX Genus Species Number Primers Results Neisseria gonorrhoeae 108CT/NG All Positive

The exclusivity (i.e., a measure of the ability to exclude falsepositives when neisserial organisms of species other than gonorrhoeaeare present in samples) of these assays is illustrated in Table X.

TABLE X Genus Species Number Primers Results Neisseria animalis 3 CT/NGAll Negative Neisseria caviae 2 ″ ″ Neisseria cinerea 6 ″ ″ Neisseriacuniculi 1 ″ ″ Neisseria denitrificans 2 ″ ″ Neisseria elongata 4 ″ ″Neisseria flava 1 ″ ″ Neisseria flavescens 7 ″ ″ Neisseria kochi 1 ″ ″Neisseria lactamica 6 ″ ″ Neisseria meningitidis 21 ″ ″ Neisseria mucosa14 ″ ″ Neisseria perflava 6 ″ ″ Neisseria polysaccharea 3 ″ ″ Neisseriasicca 7 ″ ″ Neisseria subflava 1 ″ ″ Neisseria subflava biovar flava 1 ″″ Neisseria subflava biovar perflava 2 ″ ″ Neisseria subflava biovar 2 ″″ subflava/flava Neisseria subflava perflava 5 ″ ″ Total 95 All Negative

The specificity (i.e., a measure of the ability to exclude falsepositives when non-neisserial organisms are present in samples) of theseassays is illustrated in Table XI.

TABLE XI Genus Species Number Primers Results Chlamydia trachomatis 15NG All Negative Chlamydia pneumonae 1 ″ ″ Chlamydia psittaci 1 ″ ″Achromobacter xerosis 1 ″ ″ Acinetobacter lwoffi 1 ″ ″ Acinetobactercalcoaceticus 1 ″ ″ Acinetobacter sp. genospecies 3 1 ″ ″ Actinomycesisrealii 1 ″ ″ Aerococcus viridans 1 ″ ″ Aeromonas hydrophila 1 ″ ″Agrobacterium radiobacter 1 ″ ″ Alcaligenes faecalis 1 ″ ″ Bacillusthuringiensis 1 ″ ″ Bacillus subtilis 1 ″ ″ Bacteriodes fragilis 1 ″ ″Bacteroides caccae 1 ″ ″ Bifidobacillus longum 1 ″ ″ Bifidobacteriumadolescentis 1 ″ ″ Branhamella catarrhalis 1 ″ ″ Brevibacterium linens 1″ ″ Candida albicans 1 ″ ″ Chromobacter violaceum 1 ″ ″ Citrobacterfreundii 1 ″ ″ Clostridium innocuum 1 ″ ″ Clostridium perfringens 1 ″ ″Corynebacterium genitalium 1 ″ ″ Corynebacterium xerosis 1 ″ ″Cryptococcus neoformans 1 ″ ″ Deinococcus radiopugnans 1 ″ ″ Derxiagummosa 1 ″ ″ Echerichia coli 1 ″ ″ Eikenella corrodens 1 ″ ″Enterobacter cloacae 1 ″ ″ Enterococcus avium 1 ″ ″ Enterococcusfaecalis 1 ″ ″ Enterococcus faecium 1 ″ ″ Erysipelothrix rhusiopathiae 1″ ″ Ewingella americana 1 ″ ″ Flavobacterium meningosepticum 1 ″ ″Gamella haemolysans 1 ″ ″ Gamella morbillorum 1 ″ ″ Gardnerellavaginalis 1 ″ ″ Haemophilus influenzae 1 ″ ″ Haemophilus ducreyi 1 ″ ″Kingella kingae 1 ″ ″ Klebsiella pneumoniae ss ozaenae 1 ″ ″Lactobacillus oris 1 ″ ″ Lactobacillus vaginalis 1 ″ ″ Lactobacillusacidophillus 1 ″ ″ Lactobacillus brevis 1 ″ ″ Lactobacillus crisptus 1 ″″ Lactobacillus lactis lactis 1 ″ ″ Lactobacillus parabuchnerri 1 ″ ″Lactococcus lactis cremoris 1 ″ ″ Legionella bozemanii 1 ″ ″ Legionellapneumophila 1 ″ ″ Leuconostoc paramesenteroides 1 ″ ″ Micrococcus luteus1 ″ ″ Moraxella osloensis 1 ″ ″ Morganella morganii 1 ″ ″ Mycobacteriumsmegmatis 1 ″ ″ Mycoplasma hominis 1 ″ ″ Serratia denitrificans 1 ″ ″Pasteurella maltocida 1 ″ ″ Pediococcus acidilactica 1 ″ ″Peptostreptococcus magnus 1 ″ ″ Peptostreptococcus productus 1 ″ ″Prevotella bivia 1 ″ ″ Prevotella corporis 1 ″ ″ Prevotella intermedia 1″ ″ Propionibacterium acnes 1 ″ ″ Proteus mirabilis 1 ″ ″ Providenciastuartii 1 ″ ″ Pseudomonas aeruginosa 1 ″ ″ Pseudomonas putida 1 ″ ″Rahnella aquatilis 1 ″ ″ Salmonella minnesota 1 ″ ″ Salmonellatyphimurium 1 ″ ″ Serratia marscence 1 ″ ″ Staphylococcus aureus 1 ″ ″Staphylococcus epidermidis 1 ″ ″ Streptococcus salivarius 1 ″ ″Streptococcus agalactiae 1 ″ ″ Streptococcus anginosus 1 ″ ″Streptococcus bovis 1 ″ ″ Streptococcus dysgalatia 1 ″ ″ Streptococcusequinis 1 ″ ″ Streptococcus pneumoniae 1 ″ ″ Streptococcus pyogenes 1 ″″ Vibrio parahaemolyticus 1 ″ ″ Yersinia enterocolitica 1 ″ ″ Treponemapallidum 1 ″ ″ Herpes simplex virus 1 1 ″ ″ Herpes simplex virus 2 1 ″ ″Epstein Barr Virus 1 ″ ″ Human papilloma virus type 16 1 ″ ″ Humanpapilloma virus type 18 1 ″ ″ Total 111 All Negative

Example III Detection of N. gonorrhoeae Via Neisseria Gonorrhoeae DirectRepeat 33

Selection and synthesis of Neisseria gonorrhoeae Direct Repeat 33Specific Oligonucleotide Primers for PCR Analysis

The Neisseria gonorrhoeae Direct Repeat 33 (NGDR33) was previouslyidentified as a two-copy DNA sequence in the Neisseria gonorrhoeaegenome. The sequence of NGDR33 was obtained from the Los Alamos NationalLaboratory Sexually Transmitted Diseases database via the world wide webat stdgen.lanl.gov/stdgen/bacteria/ngon/. A blast homology search ofNGDR33 revealed numerous significant hits with Neisseria meningitidisand the entire 1142 base pair NGDR33 sequence has 38.09% identity withgaps (50.44% identity without gaps) to N. meningitidis serogroup Bstrain MC58 section 77 (GenBank® accession number AE002435). FIG. 8depicts a Clustal W alignment of the NGDR33 sequence (SEQ ID NO:2) witha portion of this N. meningitidis sequence (SEQ ID NO:35). The NGDR33sequence was scanned for regions of minimal sequence identity with N.meningitidis and upstream and downstream oligonucleotide primers (NG613(5′-AATGTCGGGTTTGACGAAACTC-3′ (SEQ ID NO: 15)) and a complement to NG614(i.e., 5′-AACGTCCGACAACCGGTAAC-3′ (SEQ ID NO:27)), respectively spanninga 265 base pair region of NGDR33 were synthesized. The positions ofNG613 and NG614 are underlined in FIG. 8. The deoxycytidylate units atthe 3′ ends of both of these primers had been modified, as referred toabove, to include t-butyl benzyl groups.

Neisserial Genomic DNA Purification, Amplification, and AnalyticalAgarose Gel Electrophoresis

The genomic DNA of various neisserial species was purified as describedabove in Example I. Separate PCR reactions were performed using genomicDNA isolated from various neisserial species as templates and the pairof primers, NG613 and the complement to NG614 (described above), using aprocedure similar to that described above used to amplify the 190 basepair segment of NGDR9. In addition, the PCR products of theseamplification reactions were electrophoretically separated as describedabove in Example I. The gels were examined for the presence or absenceof PCR amplification products at the expected size of 265 bp. FIGS. 9 Aand B, and 10 are photographs of agarose gels that show the detection ofthis 265 base pair segment of NGDR33

Example IV Detection of N. gonorrhoeae/C. trachomatis in ClinicalSamples

This prophetic example describes a protocol for the detection of N.gonorrhoeae and C. trachomatis in clinical samples.

Clinical Samples

Endocervical swab specimens from women and urethral swab specimens frommen are collected by standard procedures known in the art. Swabs areinoculated into suitable culture transport media (e.g., 2SP, M-4(Microtest, Inc., Atlanta, Ga.), Bartel's chlamydial (Intracel Corp.,Issaquah, Wash.), etc.), which is then used for PCR analysis (see also,Van der Pol et al. (2000) J. Clin. Microbiol. 38:1105-1112, which isincorporated by reference). These specimens are generally stored at 2 to8° C. and are typically transported to the laboratory within 24 to 72hours of collection. The specimens are typically vortexed with the swabstill in the tube, cell cultures are inoculated, and an aliquot of eachspecimen is transferred to a new tube, which is generally stored at 2 to8° C. for up to 7 days postcollection and then processed for PCRanalysis.

Optionally, 50 ml aliquots of first-catch urine is also collected fromboth men and women. Female urine specimens are collected either beforeor after swab collection. Male urine specimens are collected after theurethral swab specimens have been obtained. Urine specimens aretypically stored at room temperature and transported to the laboratorywithin 24 hours or are stored at, e.g., 2 to 8° C. if not transportedwithin 24 hours of collection. Upon arrival at the laboratory, a 500 μlaliquot is typically stored at 2 to 8° C. for up to 7 days from the timeof collection until it is processed for PCR analysis.

PCR Analysis

Each specimen is typically processed and subjected to either or both theAMPLICOR® and COBAS AMPLICOR® tests as described in the manufacturer'spackage inserts. For each processed specimen, the C. trachomatis, N.gonorrhoeae, and internal control (IC) target DNAs are simultaneouslyamplified in a single reaction mixture that contains at least two primerpairs, at least one pair specific for C. trachomatis and at least onepair specific for N. gonorrhoeae (e.g., comprising at least one sequenceselected from SEQ ID NOS: 3-27). The resulting amplification productsare generally captured separately and detected colorimetrically byhybridization to microwell plates (AMPLICOR® format) (Crotchfelt et al.(1997) “Detection of Neisseria gonorrhoeae and Chlamydia trachomatis ingenitourinary specimens from men and women by a coamplification PCRassay,” J. Clin. Microbiol. 35:1536-1540, which is incorporated byreference) or to magnetic microparticles (COBAS AMPLICOR® format) coatedwith N. gonorrhoeae- (e.g., comprising sequences selected from SEQ IDNOS: 3-27), C. trachomatis-, and IC-specific oligonucleotide probes. TheCOBAS AMPLICOR® analyzer automatically performs all of theamplification, hybridization, and detection steps (DiDomenico et al.(1996) “COBAS AMPLICOR™: a fully automated RNA and DNA amplification anddetection system for routine diagnostic PCR,” Clin. Chem. 42:1915-1923,Jungkind et al. (1996) “Evaluation of automated COBAS AMPLICOR PCRsystem for detection of several infectious agents and its impact onlaboratory management,” J. Clin. Microbiol. 34:2778-2783, which are bothincorporated by reference). In the AMPLICOR® format, amplification isperformed, e.g., with a GeneAmp® PCR System 9700 thermal cycler(Perkin-Elmer, Norwalk, Conn.) and hybridization and detection areperformed manually (Loeffelholz et al. (1992) “Detection of Chlamydiatrachomatis in endocervical specimens by polymerase chain reaction,” J.Clin. Microbiol. 30:2847-2851, which is incorporated by reference).

Data Analysis

Specimens yielding signals above a positive cutoff (optical density [OD]of 2.0 (A₆₆₀) for C. trachomatis and OD of 3.5 (A₆₆₀) for N.gonorrhoeae) are typically interpreted as positive for the particularorganism, regardless of the IC result. Specimens yielding N. gonorrhoeaeor C. trachomatis signals below a negative cutoff (e.g., OD of 0.2) aretypically interpreted as negative for the particular organism, providedthat the IC signal is above the assigned cutoff (e.g., OD of 0.2) andthe test considered valid. Specimens yielding N. gonorrhoeae or C.trachomatis signals below the cutoff values for both N. gonorrhoeae orC. trachomatis and IC are generally interpreted as inhibitory.Inhibitory specimens are typically retested by processing of a frozenaliquot of the original specimen. The repeat test results are classifiedusing the above criteria.

Specimens yielding results between the negative and positive cutoffs(≧0.2, <3.5 for N. gonorrhoeae and ≧0.2, <2.0 for C. trachomatis) aretypically considered equivocal for the particular organism, regardlessof the IC signal. Equivocal results are generally resolved by processingan aliquot of the original specimen, retesting in duplicate andcomparing the results to the initial test. These specimens are typicallyinterpreted as positive for the particular organism if at least twovalid tests yield an N. gonorrhoeae or C. trachomatis OD of ≧2.0 for theparticular organism. These specimens are generally interpreted asnegative for the particular organism if the two repeat tests yield N.gonorrhoeae or C. trachomatis signals of <0.2 OD for the particularorganism, provided that the IC signals are above the assigned cutoff. Ifthe two repeat tests yield an N. gonorrhoeae or C. trachomatis OD of<0.2 for the particular organism and the IC signal is below the assignedcutoff for either of the duplicate repeat tests, the specimen isgenerally interpreted as inhibitory.

Example V Detection of N. gonorrhoeae Via Neisseria gonorrhoeae DirectRepeat 9 Variant

Selection and synthesis of NGDR9Var oligonucleotides for PCR analysis

As described in Example I, the Neisseria gonorrhoeae Direct Repeat 9(NGDR9) previously was identified as a two-copy DNA sequence in theNeisseria gonorrhoeae genome. It was subsequently confirmed as a 3-copyDNA sequence. However, during inclusivity testing of a prototype NGassay with NGDR9 primers, 4 N. gonorrhoeae strains (including strainNG889) isolated from clinical samples, were not amplified. Subsequentsequence analysis of genomic DNA from strain NG889 revealed the presenceof 3 identical copies of a variant sequence (NGDR9Var), with significantmismatches within the target region of NGDR9 that precludedamplification by the NG519 (SEQ ID NO: 5) and NG514 (SEQ ID NO: 24)primers. The entire 727 base pair NGDR9Var sequence (SEQ ID NO: 36)bears no identity with any sequence in the Neisseria meningitidis genomebut has 70% overall identity with the NGDR9 sequence (SEQ ID NO: 1). TheNGDR9Var sequence was scanned for maximum sequence identity with variantsequences from additional N. gonorrhoeae strains, and upstream anddownstream oligonucleotide primers NG579 (5′-GTTTCGACAGGCTTGCCAA-3′)(SEQ ID NO: 38) and NG552 (5′-CCTGTTTGCGACAAAGAGCA-3′) (SEQ ID NO: 40)spanning a 215 bp region of NGDR9Var were synthesized. Thedeoxyadenosine base at the 3′ ends of both of these primers had beenmodified to include t-benzyl groups as described in, e.g., U.S. Pat. No.6,001,611, entitled “MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS,”issued Dec. 14, 1999 to Will, which is incorporated by reference. Thepositions of NG579 and NG552 in NGDR9Var are underlined in FIG. 11.

Amplification of 215 bp Segment of NGDR9Var

Separate PCR reactions were performed using genomic DNA isolated fromthe various neisserial species (as described in Example I) as templatesand the pair of primers NG579 and NG552 (described above). PCRs wereperformed in volumes of 100 μl containing 50 mM Tricine (pH 8.3), 80 mMK(OAc)₂ (pH 7.5), 1 mM Mn(OAc)₂ (pH 6.5), 5% glycerol, 1 mM Mg(OAc)₂ (pH6.5), 50 μM dATP, 50 μM dGTP, 50 μM dCTP, 100 μM dUTP, 20 U of ZO5® DNApolymerase (Roche Molecular Systems, Alameda, Calif.), 5U AmpErase® UNG(Uracil-N-Glycosylase), and 0.5 μM of each primer. Genomic DNA was addedas template at 10³ genomic equivalents per reaction for Neisseriagonorrhoeae and 10⁶ genomic equivalents per reaction for Neisseriameningitidis and other neisserial strains. Reactions were performed for60 cycles of denaturation at 95° C. for 15 sec, annealing at 58° C. for40 sec, and a final extension at 72° C. for 5 min using a COBAS Taqman®PCR System (Roche Molecular Systems, Alameda, Calif.).

Analytical Agarose Gel Electrophoresis of NGDR9Var PCR Products

The PCR products were prepared for gel electrophoresis analysis asdescribed in Example I. The gels were examined for the presence orabsence of PCR amplification products at the expected size of 215 bp.

FIGS. 12A and B are photographs of agarose gels that show the detectionof the 215 base pair segment of NGDR9Var.

Example VI Detection of N. Gonorrhoeae Via Neisseria Gonorrhoeae DirectRepeat 9

Selection and Synthesis of NGDR9Univ Oligonucleotides for PCR Analysis

In order to utilize a single set of oligonucleotides for simultaneousamplification and detection of NGDR9 and NGDR9Var sequences, bothsequences were scanned for regions of maximum sequence identity andconsensus sequence upstream and downstream oligonucleotides NGSU2(5′-GCGGCAAGCATCTGTTTTGC-3′) (SEQ ID NO: 47) and NGSL2(5′-AGTAGCAGGCGCGAAGATTGA-3′) (SEQ ID NO: 49) spanning a 473 base pairregion of NGDR9 and a 394 base pair region of NGDR9Var were synthesized.The deoxycytosine and deoxyadenosine bases at the 3′ ends of NGSU2 andNGSL2 primers respectively, had been modified to include t-benzyl groupsas described in, e.g., U.S. Pat. No. 6,001,611, entitled “MODIFIEDNUCLEIC ACID AMPLIFICATION PRIMERS,” issued Dec. 14, 1999 to Will, whichis incorporated by reference. The positions of NGSU2 and NGSL2 in NGDR9and NGDR9Var are underlined in FIG. 11.

Amplification of 473 bp Segment of NGDR9 and 394 bp of NGDR9Var

Separate PCR reactions were performed using genomic DNA isolated fromthe various neisserial species (as described in Example I) as templatesand the pair of primers NGSU2 and NGSL2 (described above). PCRs wereperformed in volumes of 100 μl containing 50 mM Tricine (pH 8.3), 80 mMK(OAc)₂ (pH 7.5), 1 mM Mn(OAc)₂ (pH 6.5), 1 mM Mg(OAc)₂ (pH 6.5), 5%glycerol, 50 μM dATP, 50 μM dGTP, 50 μM dCTP, 100 μM dUTP, 20 U of ZO5®DNA polymerase (Roche Molecular Systems, Alameda, Calif.), 5U AmpErase®UNG (Uracil-N-Glycosylase), and 0.5 μM of each primer. Genomic DNA wasadded as template at 10³ genomic equivalents per reaction for Neisseriagonorrhoeae and 10⁶ genomic equivalents per reaction for Neisseriameningitidis and other neisserial strains. Reactions were performed for60 cycles of denaturation at 95° C. for 15 sec, annealing at 58° C. for40 sec, and a final extension at 72° C. for 5 min using a COBAS Taqman®PCR System (Roche Molecular Systems, Alameda, Calif.).

Analytical Agarose Gel Electrophoresis of NGDR9Univ PCR Products

The PCR products were prepared for gel electrophoresis analysis asdescribed in Example I. The gels were examined for the presence orabsence of PCR amplification products at the expected sizes of 473 bpfor the NGDR9 and 394 bp for the NGDR9Var.

FIGS. 13A and B are photographs of agarose gels that show the detectionof the 473 base pair segment of NGDR9 and the 394 base pair segment ofNGDR9Var.

Example VII Assays Illustrating the Selective Detection of N.gonorrhoeae with NGDR9Var Primers

This example provides lists of organisms that were analyzed in assaysthat included the use of primer nucleic acids NG579 (SEQ ID NO: 38) andNG552 (SEQ ID NO: 40) and a 5′-nuclease probe (having a sequencecorresponding to SEQ ID NO: 42) alone or in combination with C.trachomatis primers.

The inclusivity (i.e., a measure of the ability to detect the targetorganism, N. gonorrhoeae in samples) of these assays is illustrated inTable XII.

TABLE XII Number Number Genus Species tested Primers Positive Neisseriagonorrhoeae 538 NGDR9Var 62

The exclusivity (i.e., a measure of the ability to exclude falsepositives when neisserial organisms of species other than gonorrhoeaeare present in samples) of these assays is illustrated in Table XIII.

TABLE XIII Genus Species Number Primers Results Neisseria animalis 3NGDR9Var Negative Neisseria caviae 2 ″ ″ Neisseria cinerea 5 ″ ″Neisseria cuniculi 1 ″ ″ Neisseria denitrificans 2 ″ ″ Neisseriaelongata 5 ″ ″ Neisseria flava 1 ″ ″ Neisseria flavescens 7 ″ ″Neisseria kochi 1 ″ ″ Neisseria lactamica 6 ″ ″ Neisseria meningitidis24 ″ ″ Neisseria mucosa 14 ″ ″ Neisseria perflava 6 ″ ″ Neisseriapolysaccharea 3 ″ ″ Neisseria sicca 7 ″ ″ Neisseria subflava 1 ″ ″Neisseria subflava biovar flava 1 ″ ″ Neisseria subflava biovar 3 ″ ″subflava/flava Neisseria subflava perflava 7 ″ ″ Total 99

The specificity (i.e., a measure of the ability to exclude falsepositives when non-neisserial organisms are present in samples) of theseassays is illustrated in Table XIV.

TABLE XIV Genus species Number Primers Results Chlamydia trachomatis 15NGDR9Var Negative Chlamydia pneumoniae 1 ″ ″ Chlamydia psittaci 1 ″ ″Achromobacter Xerosis 1 ″ ″ Acinetobacter Lwoffi 1 ″ ″ Acinetobactercalcoaceticus 1 ″ ″ Actinomyces isrealii 1 ″ ″ Aerococcus viridans 1 ″ ″Aeromonas hydrophila 1 ″ ″ Agrobacterium radiobacter 1 ″ ″ Alcaligenesfaecalis 1 ″ ″ Bacillus thuringiensis 1 ″ ″ Bacillus subtilis 1 ″ ″Bacteriodes fragilis 1 ″ ″ Bacteroides caccae 1 ″ ″ Bifidobacilluslongum 1 ″ ″ Bifidobacterium adolescentis 1 ″ ″ Branhamella catarrhalis1 ″ ″ Brevibacterium linens 1 ″ ″ Candida albicans 1 ″ ″ Chromobacterviolaceum 1 ″ ″ Citrobacter freundii 1 ″ ″ Clostridium innocuum 1 ″ ″Clostridium perfringens 1 ″ ″ Corynebacterium genitalium 1 ″ ″Corynebacterium xerosis 1 ″ ″ Cryptococcus neoformans 1 ″ ″ Echerichiacoli 1 ″ ″ Eikenella corrodens 1 ″ ″ Enterobacter cloacae 1 ″ ″Enterococcus avium 1 ″ ″ Enterococcus faecalis 1 ″ ″ Enterococcusfaecium 1 ″ ″ Erysipelothrix rhusiopathiae 1 ″ ″ Ewingella americana 1 ″″ Flavobacterium meningosepticum 1 ″ ″ Gamella morbillorum 1 ″ ″Gardnerella vaginalis 1 ″ ″ Haemophilus influenzae 1 ″ ″ Haemophilusducreyi 1 ″ ″ Kingella kingae 1 ″ ″ Klebsiella pneumoniae ss ozaenae 1 ″″ Lactobacillus oris 1 ″ ″ Lactobacillus vaginalis 1 ″ ″ Lactobacillusacidophillus 1 ″ ″ Lactobacillus brevis 1 ″ ″ Lactobacillusparabuchnerri 1 ″ ″ Legionella bozemanii 1 ″ ″ Legionella pneumophila 1″ ″ Leuconostoc paramesenteroides 1 ″ ″ Micrococcus luteus 1 ″ ″Moraxella osloensis 1 ″ ″ Morganella morganii 1 ″ ″ Mycobacteriumsmegmatis 1 ″ ″ Mycoplasma hominis 1 ″ ″ Serratia denitrificans 1 ″ ″Pasteurella maltocida 1 ″ ″ Peptostreptococcus magnus 1 ″ ″Peptostreptococcus productus 1 ″ ″ Prevotella bivia 1 ″ ″ Prevotellacorporis 1 ″ ″ Prevotella intermedia 1 ″ ″ Propionibacterium acnes 1 ″ ″Proteus mirabilis 1 ″ ″ Providencia stuartii 1 ″ ″ Pseudomonasaeruginosa 1 ″ ″ Pseudomonas putida 1 ″ ″ Salmonella minnesota 1 ″ ″Salmonella typhimurium 1 ″ ″ Serratia marcescens 1 ″ ″ Staphylococcusaureus 1 ″ ″ Staphylococcus epidermidis 1 ″ ″ Streptococcus salivarius 1″ ″ Streptococcus agalactiae 1 ″ ″ Streptococcus bovis 1 ″ ″Streptococcus dysgalatia 1 ″ ″ Streptococcus equis 1 ″ ″ Streptococcuspneumoniae 1 ″ ″ Streptococcus pyogenes 1 ″ ″ Vibrio parahaemolyticus 1″ ″ Yersinia enterocolitica 1 ″ ″ Treponema pallidum 1 ″ ″ Herpessimplex virus 1 1 ″ ″ Herpes simplex virus 2 1 ″ ″ Epstein Barr Virus 1″ ″ Human papilloma virus type 16 1 ″ ″ Human papilloma virus type 18 1″ ″ Total 100

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. An oligonucleotide comprising a nucleic acid having at least 90%sequence identity to one of SEQ ID NOS: 37-60 or a complement thereof,which oligonucleotide has 50 or fewer nucleotides.
 2. Theoligonucleotide of claim 1, wherein the nucleic acid has at least 95%sequence identity to one of SEQ ID NOS: 37-60 or the complement thereof.3. The oligonucleotide of claim 1, wherein the oligonucleotide comprisesat least one modified nucleotide.
 4. The oligonucleotide of claim 1,wherein the oligonucleotide comprises at least one label and/or at leastone quencher moiety.
 5. The oligonucleotide of claim 1: wherein theoligonucleotide has 40 or fewer nucleotides.
 6. The oligonucleotide ofclaim 1, wherein the oligonucleotide comprises at least oneconservatively modified variation.
 7. A method of detecting Neisseriagonorrhoeae in a sample, the method comprising: (a) contacting nucleicacids from the sample with at least a first pair of primer nucleic acidscomprising at least one nucleic acid selected from the group consistingof: SEQ ID NOS: 37-60, a substantially identical variant thereof whereinthe variant has at least 90% sequence identity to one of SEQ ID NOS:37-60, and complements of SEQ ID NOS: 37-60 and the variant, in at leastone nucleic acid amplification reaction; and, (b) detecting the nucleicacids and/or one or more amplicons thereof from the nucleic acidamplification reaction during or after (a), thereby detecting theNeisseria gonorrhoeae in the sample.
 8. The method of claim 7, wherein(a) comprises contacting the nucleic acids from the sample with at leasta second pair of primer nucleic acids that are at least partiallycomplementary to a Chlamydia trachomatis nucleic acid and (b) comprisesdetecting one or more additional amplicons from the nucleic acidamplification reaction during or after (a), thereby detecting Chlamydiatrachomatis in the sample.
 9. The method of claim 7, wherein at leastone of the primer nucleic acids comprises a modified primer nucleicacid.
 10. The method of claim 7, wherein at least one of the primernucleic acids comprises at least one label.
 11. The method of claim 10,wherein (b) comprises detecting a detectable signal produced by thelabel, or amplifying a detectable signal produced by the label toproduce an amplified signal and detecting the amplified signal.
 12. Themethod of claim 7, wherein (b) comprises monitoring binding between theamplicons and one or more nucleic acid detection reagents thatdetectably bind to a nucleic acid with a sequence consisting of SEQ IDNO: 36, a substantially identical variant thereof wherein the varianthas at least 90% sequence identity to one of SEQ ID NO: 36, or acomplement of SEQ ID NO: 36 or the variant.
 13. The method of claim 12,wherein at least one of the nucleic acid detection reagents comprises anucleic acid having a sequence selected from the group consisting of:SEQ ID NOS: 37-60, a substantially identical variant thereof wherein thevariant has at least 90% sequence identity to one of SEQ ID NOS: 37-60,and complements of SEQ ID NOS: 37-60 and the variant.
 14. The method ofclaim 12, wherein at least one of the nucleic acid detection reagentscomprises an oligonucleotide.
 15. The method of claim 12, wherein atleast one of the nucleic acid detection reagents comprises at least onelabel and/or at least one quencher moiety.
 16. A method of detectingNeisseria gonorrhoeae in a sample, the method comprising: (a) contactingnucleic acids from the sample with at least a first pair of primernucleic acids in at least one nucleic acid amplification reaction,wherein each of the primer nucleic acids have between 12 and 100nucleotides, and wherein at least one of the primer nucleic acidscomprises at least 90% sequence identity with a subsequence of SEQ IDNO: 36 or a complement thereof; and, (b) detecting the nucleic acidsand/or one or more amplicons thereof from the nucleic acid amplificationreaction during or after (a), thereby detecting the Neisseriagonorrhoeae in the sample.
 17. The method of claim 16, wherein (a)comprises contacting the nucleic acids from the sample with at least asecond pair of primer nucleic acids that are at least partiallycomplementary to a Chlamydia trachomatis nucleic acid and (b) comprisesdetecting one or more additional amplicons from the nucleic acidamplification reaction during or after (a), thereby detecting Chlamydiatrachomatis in the sample.
 18. The method of claim 16, wherein at leastone of the primer nucleic acids comprises a modified primer nucleicacid.
 19. The method of claim 16, wherein (b) comprises monitoringbinding between the amplicons and one or more nucleic acid detectionreagents that detectably bind to a nucleic acid with a sequenceconsisting of SEQ ID NO: 36, a substantially identical variant thereofwherein the variant has at least 90% sequence identity to SEQ ID NO: 36,or a complement of SEQ ID NO: 36 or the variant.
 20. The method of claim16, wherein one or more of the primer nucleic acids has a sequenceselected from the group consisting of: SEQ ID NOS: 37-60, asubstantially identical variant thereof wherein the variant has at least90% sequence identity to one of SEQ ID NOS: 37-60, and complements ofSEQ ID NOS: 37-60 and the variant.
 21. A kit, comprising: (a) at leastone oligonucleotide which has 50 or fewer nucleotides, whicholigonucleotide comprises at least 90% sequence identity with asubsequence of SEQ ID NO: 36 or a complement thereof; and one or moreof: (b) instructions for determining a presence of Neisseria gonorrhoeaein a sample by monitoring binding between nucleic acids and/or ampliconsthereof from the sample and the oligonucleotide, wherein the presence ofNeisseria gonorrhoeae in the sample is unknown or unsubstantiated; or,(c) at least one container for packaging at least the oligonucleotide.22. The kit of claim 21, wherein the oligonucleotide has a sequenceselected from the group consisting of: SEQ ID NOS: 37-60, asubstantially identical variant thereof wherein the variant has at least90% sequence identity to one of SEQ ID NOS: 37-60, and complements ofSEQ ID NOS: 37-60 and the variant.
 23. The kit of claim 22, furthercomprising one or more nucleic acid detection reagents that detectablybind to a Chlamydia trachomatis nucleic acid.
 24. The kit of claim 23,further comprising at least one enzyme.
 25. A reaction mixturecomprising a set of amplicons having sequences that correspond to one ormore subsequences of SEQ ID NO: 36, a substantially identical variantthereof wherein the variant has at least 90% sequence identity to SEQ IDNOS: 36, or a complement of SEQ ID NO: 36 or the variant, whichamplicons lack terminator nucleotides, wherein at least a subset of theset of amplicons is produced using at least one primer nucleic acidhaving a sequence selected from the group consisting of: SEQ ID NOS:37-60, a substantially identical variant thereof wherein the variant hasat least 90% sequence identity to one of SEQ ID NOS: 37-60, andcomplements of SEQ ID NOS: 37-60 and the variant.